Device and method for particle complex handling

ABSTRACT

An embodiment of the invention relates to a device for detecting an analyte in a sample. The device comprises a fluidic network and an integrated circuitry component. The fluidic network comprises a sample zone, a cleaning zone and a detection zone. The fluidic network contains a magnetic particle and/or a signal particle. A sample containing an analyte is introduced, and the analyte interacts with the magnetic particle and/or the signal particle through affinity agents. A microcoil array or a mechanically movable permanent magnet is functionally coupled to the fluidic network, which are activatable to generate a magnetic field within a portion of the fluidic network, and move the magnetic particle from the sample zone to the detection zone. A detection element is present which detects optical or electrical signals from the signal particle, thus indicating the presence of the analyte.

RELATED APPLICATIONS

This application is related to “ENZYMATIC SIGNAL GENERATION ANDDETECTION OF BINDING COMPLEXES IN STATIONARY FLUIDIC CHIP” (U.S. Pat.No. 8,409,877, issued Apr. 2, 2013), “METHOD AND DEVICE FOR BIOMOLECULEPREPARATION AND DETECTION USING MAGNETIC ARRAY” (U.S. Pat. No.8,586,385, issued Nov. 19, 2013), and “PROGRAMMABLE ELECTROMAGNETICARRAY FOR MOLECULE TRANSPORT” (U.S. Pat. No. 7,820,454, issued Oct. 26,2010), which are incorporated herein by reference.

FIELD OF INVENTION

The embodiments of the invention relate to devices for conductingbiomedical assays, methods of making such devices, and methods ofdetecting the presence of an analyte using such devices. Morespecifically, the embodiments relate to devices and methods that combinefluidic or fluid handling devices and magnetic microarrays with anintegrated circuitry element that perform versatile and/or convenientanalysis of an analyte with design flexibility. The invention transcendsseveral scientific disciplines such as biochemistry and physics,engineering, microelectronics, microelectromechanical systems (MEMS),analytical chemistry, and biomedical diagnostics.

BACKGROUND

Chemical analysis and biomedical diagnostics commonly use absorption,fluorescence, chemiluminescence, UV-Vis and Raman scattering to detectthe presence of an analyte. For example, enzyme-linked immunosorbentassays (ELISA) are widely used to detect an analyte. ELISA assays aretypically performed in microwell plates, and require multiple steps ofadding reagents, washing the reactant plates, and applying a reactionsubstrate that is converted to provide a chromogenic (colorimetric) orfluorescent signal. Furthermore, its detection limit ranges from themicromolar to picomolar or nanograms to pictograms of protein analyteper milliliter of a sample. For markers with low copy numbers, moresensitive detection technology is needed.

The current methods and devices for detecting the presence of an analytein a sample have multiple drawbacks. First, the sizes of the devices aretoo big to be used in field applications or at home environment, such aspoint-of-care (POC) environment. Second, the current devices require alarge amount of sample, which not only is infeasible for certainapplications, but also hinders activities such as mixing and heating ofthe sample required for many analyses. Third, the current devices havecomplex structures for fluidic control and are often not self-contained.Fourth, current devices are limited by their detection sensitivity.Fifth, the current devices are often designed for specializedapplications, e.g. protein analysis only, or nucleic acid only. Thus,there is a need for miniaturized, integrated, and versatile devices foranalysis of a sample suspected of containing an analyte that can performon-site, flexible, rapid, sensitive, and/or efficient analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an embodiment of the invention that comprises afluidic network having a sample zone and other fluidic zones, associatedwith a magnetic microcoil array, a detection element and an integratedcircuitry component, linked to a circuit board.

FIG. 2 illustrates an exemplary top-down view of a magnetic microcoilarray showing the movement of magnetic particles.

FIG. 3 illustrates a top-down view and cross-section view of the fluidicnetwork.

FIG. 4 illustrates a more detailed top-down view and cross-section viewof the fluidic network.

FIG. 5 illustrates a cross-section view of the fluidic network showingthe movement of the binding complex to the detection zone.

FIG. 6A illustrates the use of the fluidic network in moving magneticparticles. FIG. 6B illustrates the fluorescence of a mixture of magneticparticles and Qdots, before and after washing. FIG. 6C quantifies thefluorescence of the samples in the tubes from FIG. 6B and samples takenfrom the fluidic network during FIG. 6A. FIG. 6D quantifies on-chipchemiluminescent detection of PSA.

FIG. 7 illustrates the formation of sandwich binding complexes(“sandwich binding” and “tandem binding”) and a competitive bindingcomplex.

FIG. 8 illustrates the use of codes with a magnetic affinity complex anda magnetic signal affinity complex.

FIG. 9 illustrates an embodiment of the particle (or molecule) transportdevice of the invention, showing major components which are (1) afluidic network, e.g., a biochip, (2) an electromagnetic array, (3) acircuitry board; and (4) computer.

FIG. 10 illustrates electromagnetic coils of the array, showingreversible magnetic polarities, magnetic field gradients (flux)controlled by current flow directions and strength, and fluxdistributions that can be optimized by varying geometry (shapes) of theheads of the cores.

FIGS. 11A and B illustrate a power delivery system, showing an exampleof ways to minimize the number of power switches.

FIG. 12 illustrates an experiment demonstrating magnetic particleconcentrating, transporting by varying magnetic fields.

FIG. 13 illustrates a prototype system, showing the coil (inductor)array, switches and other electronic control elements, together with aprototype fluidic chip (biochip).

FIG. 14 illustrates the data and specification of a prototype system,indicating magnetic coil structure and magnetic field strengthsrelatively to coil head surface.

DETAILED DESCRIPTION

The embodiments of the invention relate to a device for detecting thepresence of an analyte in a sample. The device comprises a fluidicnetwork and an integrated circuitry component, functionally coupled to amagnetic microcoil array, a detection element, a circuit board, andoptionally, a vibration element. Specifically, the fluidic networkcomprises a plurality of fluidic zones, where each zone is connected tothe adjacent zone by a diffusion barrier. Typically one or more of thefluidic zones contains a magnetic particle and/or a signal particle. Asample suspected of containing an analyte is introduced into a fluidiczone. The analyte interacts with a magnetic particle and/or a signalparticle to form a binding complex. The magnetic microcoil array isactivated to generate a magnetic field across at least a portion of afluidic zone to separate magnetic particles and binding complexes fromuncomplexed signal particles and to move the binding complexes to afluidic zone where it can be detected by the detection element. Thedevice may further comprise servo-mechanical components and mechanismsto control the locations and movements of the detection element, themagnetic microcoil array, and optionally, a flow controller.

The embodiments of the invention also relate to methods of detecting thepresence of an analyte in a sample using the device and to methods ofmaking the device. The detection element of the embodiments of theinvention may be part of an integrated device that also serves as amicroarray or macroarray, containing an integrated circuitry component,or a microfluidic device, a MEMS, or a combination thereof. Therefore,samples contained or processed by the device may be also analyzed by thedetection element and/or the detection signals processed for analysis.If necessary, the signals determined by the detection element may betransmitted to another device for further analysis.

The embodiments of the invention relate to a device and method totransport particle complexes from one solution zone to another of aself-contained fluidic device without active liquid movement. It alsoallows for effective reactions along the fluidic channel networkinvolving concentrating, dispersing and transporting particles of microor nano dimensions. The device comprises the following major components:a) electromagnetic array, b) a set of vibrational elements, and c) acircuitry for electronic control and regulation of the magnetic array,the vibrational elements and data collection elements.

The embodiments of the invention address the problem of molecular orparticulate transport in one fluidic zone to another in a fluidic systemwithout active fluidic movement or using fluid as the transport carrier.Presently the problem described above is solved by method in whichmolecules are physically separated from one solution phase before beingplaced in another solution phase; or the molecules are immobilized onsolid surfaces and a new solution is introduced involving active fluidicmovement. The technical advantage of the embodiments of the invention isthat it allows molecular or particulate transport in one fluidic zone toanother in a fluidic system without active fluidic movement or usingfluid as the molecular transport carrier, which avoids mechanicalstructures to generate hydraulic pressure or flow, enabling simple andreliable biomedical diagnostic devices. The mixing of reagents from oneregion with those of another due to diffusion can be reduced bygeometric considerations to the level where no mechanical valves areneeded to avoid unwanted diffusional mixing, or the hydrophilic reagentsolution “droplets” are physically suspended in hydrophobic liquid suchas silicone oils through hydrophobic-hydrophilic interactions.

The embodiments of the invention relate to a device for particle complextransport and detection comprising (1) an array of electromagnetic coilswherein the coil has a magnetizable or high magnetic permeability metalcore, and the current for the coil can be controlled and varied in timeindividually, as well as reversed, to generate a specific magnetic fluxdistribution and gradient; there can be a magnetizable or high magneticpermeability coupling shape at the end of each coil whose geometry issuch to create an optimal magnetic flux, intensity and gradient, in theregion of interest; and the device is functionally coupled to a fluidicdevice to concentrate, disperse and transport particle complexes; (2) Adetection system, of optical or electrical nature; for optical: anoptical detection system consisting of a lens system and photo-diode,phototube or CCD sensing element, optionally, an optical illuminationsystem consisting of a photo-diode, LED (light emitting diodes), laseror lamp, and a spectroscopy system which could contain diachronic mirroror lens; for electrical detection, the methods can be FET detection,capacitor detection, current and voltage detection; and (3) a centralprocessing unit (CPU) functionally coupled to the array and data/signalcollection elements (optical or electrical). The device of theembodiments of the invention could optionally have the followingelements: (1) A set of magnetizable material shapes which are free to bemoved by the magnetic field generated by the electromagnets and therebyalter the magnetic field. For example, a set of magnetic high mu metalor permanent magnetic objects that can be moved by poweringelectromagnets. The movement and placement of these objects will changethe field enhancing and depleting it where needed. These magnetic shapescan be part of the array (coil cores), around the array (between arrayand fluidic device) or part of the fluidic device. (2) A set ofvibrational elements, functionally coupled to a fluidic device todisperse particle complexes, preferably, being electro-piezo vibration,or ultrasounds, wherein one or more vibrational elements could beaddressable individually. (3) A set of temperature controlling elements.For example, a set of element that can change the temperature of thefluidic device. Heating by inductively driving current in coils thefluidic device using the inductor array or electronic peltier devices.(4) A programmable system that can control the electromagnetic array ina set time sequence as well as vary the sequence depending on input fromsensing elements. Controlling the vibrational or mixing elements, thetemperature controlling elements and optical elements. Monitoringparticle complexes within the fluidic device magnetically and optically.(5) A system to move the alignment of the fluidic device relative to themagnetic array and optical and vibrational or mixing elements.

As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “an array” may include a plurality ofarrays unless the context clearly dictates otherwise.

As used herein, “magnetic particle” refers to a paramagnetic orsuperparamagnetic particle having any shape, e.g. it can have the formof a sphere, a cylinder, a cube, an oval etc., or may have a variableshape. Different types of magnetic particles which can be used with thepresent invention are described, for example, by Urs Hafeli et al. in“Scientific and Clinical Applications of Magnetic Carriers”, PlenumPress, New York, 1597, ISBN 0-306-45687-7. In one embodiment themagnetic particle comprises a streptavidin-coated magnetic bead. Themagnetic particles can be quite small, having at least one dimensionranging between 0.1 nm and 10,000 nm, preferably between 3 nm and 500nm, and more preferably between 10 nm and 300 nm. The magnetic particlescan acquire a magnetic moment due to an applied magnetic field (e.g.,they can be paramagnetic) or they can have a permanent magnetic moment.The magnetic particles can be a composite, e.g., consist of one or moresmall magnetic particles inside or attached to a non-magnetic material,or a hetero/hybrid nanostructures, such as dumbbell-shaped magnetic goldnanoparticles, which are composed by one half of magnetic particle andanother half of gold nanoparticle. As such, the term “magnetic particle”encompasses magnetic affinity complexes, coded magnetic affinitycomplexes, hybrid magnetic complexes, and coded magnetic signal affinitycomplexes, among others. In certain embodiments, the sample zone of thefluidic device comprises the magnetic particle. In other embodiments,different or the same magnetic particles can be contained within morethan one fluidic zone.

As used herein, a “magnetic affinity complex” comprises a magneticparticle functionally coupled to an affinity agent. The term “affinityagent” generally refers to a molecule that binds to an analyte for thedetection and/or analysis of the analyte and is described in more detailbelow. Non-limiting examples of affinity agents include example ofaffinity agents including antibodies, lectins, streptavidin,oligonucleotides, peptides, and oligosaccharides. It can be coupled tothe magnetic particle using a functionalized polymer, for example.

A “coded magnetic affinity complex” comprises a magnetic particlefunctionally coupled to an affinity agent and to a code. A “code” is arecognizable structure/molecule such as a polynucleotide sequence thatcorrelates to the affinity agent and thus can be used to identify orquantify the analyte.

A “coded magnetic signal affinity complex” comprises a magnetic signalparticle functionally coupled to an affinity agent and to a code. A“magnetic signal particle” is a nanoparticle having magnetic propertiesthat is detectable by the detection element of the fluidic device. Itcan be detected by various means, including electrical sensing methods(i.e., FET), optical methods (UV-Vis, IR, Raman, fluorescence,chemiluminescence, evanescence, surface plasmon), magnetic imagingmethods (such as MRI), enzymatic methods (production of a reactionproduct due to the interaction of a catalytic element with a reactionsubstrate, or alternatively, the amplification of a polynucleotide byPCR), and non-enzymatic chemical amplification methods.

A “signal particle” is a nanoparticle that is detectable by thedetection element of the device, and thus encompasses signal affinitycomplexes, signal analyte complexes, and coded magnetic signal affinitycomplexes, among others. In certain embodiments the signal particle is asurface-enhanced Raman spectroscopy (SERS)-active nanoparticle, afluorescent nanoparticle, a nanoparticle coupled to a surface-enhancedfluorescent tag, a nanoparticle containing contrast reagents, or a corenanoparticle covalently coupled to a catalytic element. In oneembodiment, the signal particle is a COIN (composite organic-inorganicnanoparticles) particle. In other embodiments, the signal particle is aQdot (quantum dot), or another fluorescent nanoparticle, such as SEF(surface-enhanced fluorescence) nanoparticle or a FluoDot™. In furtherembodiments, the signal particle is any nanoparticle (i.e. gold, silver,CdS, CdSe, copper, Eu³⁺-coated polymer, an organic polymer (homo orhetero), polymer particles incorporated with organic dyes, an inorganiccompound, or composite compounds, etc.). Additionally, the SERS-activenanoparticle and fluorescent nanoparticle can also be functionallycoupled to a catalytic element. In certain embodiments, the sample zoneof the fluidic device comprises the signal particle. Alternatively, thesample particle is contained within another fluidic zone. In furtherembodiments, different or the same signal particles can be containedwithin more than one fluidic zone.

A “signal affinity complex” comprises a signal particle functionallycoupled to an affinity agent. A “signal analyte complex” refers to asignal particle functionally coupled to an analyte. An “analyte” refersto a molecule or biological cell of interest that is to be analyzed ordetected using the devices and methods described herein, and isdescribed further below.

A “catalytic element” is a compound that serves as an agent to cause achemical reaction to occur in a reaction substrate, where the reactionproduct is detectable by the detection element. In certain embodiments,the catalytic element is selected from the group consisting of alkalinephosphatase, horseradish peroxidase, glucose oxidase, fireflyluciferase, Renilla luciferase, bacterial luciferase, other enzymes oranalogs or combinations thereof.

The catalytic element can be conjugated to the signal particle through afunctionalized polymer. For example, a polymer with a functional group(i.e. aldehyde, amine, carboxylic acid, biotin) is used to conjugate theaffinity agent and/or catalytic element to the signal particle.Conjugation or bio-conjugation can be through non-covalent interactionssuch as hydrophobic or electrostatic interactions, or through covalentinteractions, such as amide bond formation.

The analyte interacts with the magnetic particle and/or signal particleto form a binding complex, which includes any combination of theabove-described magnetic particles and signal particles. Bindingcomplexes include for example, sandwich binding complexes, magneticbinding complexes, signal binding complexes, competitive bindingcomplexes, coded magnetic binding complexes, and coded magnetic signalbinding complexes.

A “sandwich binding complex” comprises a magnetic affinity complex, asignal affinity complex, and an analyte. For example, a sample suspectedof comprising an analyte is introduced into the sample zone of thefluidic device. The analyte interacts sequentially or simultaneouslywith a magnetic affinity complex and a signal affinity complex to form asandwich binding complex. Typically the affinity agent coupled to themagnetic particle is different than the affinity agent coupled to thesignal particle, although both are complementary to the analyte. Themicrocoil array is activated to move the sandwich binding complex to thedetection zone. Uncomplexed signal particles are left behind withoutbeing transported. The signal detected from the sandwich binding complexindicates the presence of the analyte. Typically this method is usefulfor determining the presence of proteins (including peptides, antibodiesand autoantibodies) or nucleic acids.

A “super-binding complex” comprises a magnetic affinity complex,analyte, a coded affinity complex, and a-signal affinity complex. Forexample, a sample suspected of comprising an analyte is introduced intoa fluidic zone and combined with a magnetic affinity complex to form amagnetic binding complex. The array of microcoils is activated to movethe magnetic binding complex to a zone of the fluidic network comprisinga coded affinity complex, which in one embodiment, is not magnetic. Themagnetic binding complex and the coded affinity complex form a codedsandwich binding complex. The array of microcoils is activated to movethe coded sandwich binding complex to a zone of the fluidic networkcomprising a signal affinity complex, wherein the coded sandwich bindingcomplex and signal affinity complex form a super-binding complex. Thistransport moves the coded sandwich binding complex away from the unboundcoded affinity complex. The microcoils are again activated to move thesuper-binding complex away from unbound signal affinity complex and tothe detection zone, where it is detected, and where detection of thesuper-binding complex indicates the presence of the analyte.

A “coded affinity complex” comprises a particle functionally coupled toan affinity agent and a code. It is contemplated that the particle insuch a complex may or may not be magnetic.

A “magnetic binding complex” comprises a magnetic affinity complex andan analyte.

A “signal binding complex” comprises a signal affinity complex and ananalyte.

A “competitive binding complex” comprises a magnetic affinity complexand a signal analyte complex. A competitive binding complex can beformed using the methods and devices of certain embodiments of theinvention. For example, a sample suspected of comprising an analyte isintroduced into the sample zone of the fluidic device, wherein amagnetic affinity complex binds to the analyte to form a magneticbinding complex. The microcoil array is activated to move the magneticbinding complex from the sample zone to another fluidic zone. Theanalyte is displaced from the magnetic binding complex with a signalanalyte complex. The combination of the signal analyte complex and themagnetic binding complex forms a competitive binding complex. The signaldetected from the signal analyte complex that did not form thecompetitive binding complex indicates the presence of the analyte.Typically this method is useful for determining the presence of a smallmolecule, such as, but not limited to, sugars, drugs, steroids, andvitamins. in an alternative binding scheme: a) competitive bindingcomplexes are pre-formed with magnetic affinity complex andanalyte-conjugated signal affinity complex, b) the competitive bindingcomplexes are directed to sample zone, where sample analyte displacesanalyte-conjugated signal affinity complexes, c) the magnetic bindingcomplexes are moved away from the sample zone by activating themicrocoil array, and d) displaced analyte-conjugated signal affinitycomplex are detected in the sample zone, wherein the signal strength isproportional to the amount of sample analyte.

A “coded magnetic binding complex” comprises a magnetic affinitycomplex, an analyte, and a code. A “coded magnetic signal bindingcomplex” comprises a magnetic signal affinity complex, an analyte and acode. Both of these binding complexes can be formed using the methodsand devices of certain embodiments of the invention. Typically, a samplesuspected of comprising an analyte is introduced into the sample zone ofthe fluidic device, wherein a coded magnetic affinity complex binds tothe analyte to form a coded magnetic binding complex. The microcoilarray is activated to move the coded magnetic binding complex from thesample zone to a first affinity surface, where it is bound andimmobilized. Typically the affinity agent on the first affinity surfaceis complementary to and binds to the affinity agent on the magneticparticle. The code is then detached from the coded magnetic bindingcomplex. The detached code then binds to a magnetic signal affinitycomplex to form a coded magnetic signal binding complex. Typically theaffinity agent of the magnetic signal affinity complex is complementaryto the code. In one embodiment, the affinity agent of the magneticsignal affinity complex is a polynucleotide complementary to the codepolynucleotide. The microcoil array is activated to move the codedmagnetic signal binding complex to one or multiple detection zonescomprising a second affinity surface. Typically different areas of thedetection zone or the different detection zones contain unique affinityagents to the codes. The affinity agents of the second affinity surfaceare complementary to and bind the code. The detection element thendetects the coded magnetic signal binding complex in the detection zoneusing electrical sensing methods, optical sensing methods, or enzymaticmethods, such as amplifying the affinity agent (if it is apolynucleotide) on the magnetic signal affinity complex.

It is contemplated that the analyte will bind to the affinity agentcoupled to the magnetic particle, the signal particle, and/or theaffinity surface. “Binds to” refers to the interaction of the analytewith the affinity agent, which is typically a non-covalent interaction.The interaction of the analyte with the affinity agent can becharacterized in terms of a binding affinity. Binding affinity can bereadily determined using standard technology. For example, the BIAcore™system (Uppsala, Sweden) is one method for determining binding affinity.The BIAcore™ system uses surface plasmon resonance (SPR, Welford K.1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods inEnzymology 295: 268) to monitor biomolecular interactions in real time.BIAcore™ analysis conveniently generates association rate constants,dissociation rate constants, equilibrium dissociation constants, andaffinity constants. In certain embodiments, the affinity agent binds tothe analyte with a binding affinity of at least 10³ M⁻¹, more preferablyat least 10⁵ M⁻¹, and still more preferably, at least 10⁷M⁻¹.

A “substrate” refers to a material or a combination of materials uponand/or within which other or additional materials are formed, attached,or otherwise associated with according to a predetermined fashion. Asubstrate often provides physical and functional support to the other oradditional materials such that, together, they form part or whole of afunctional device. A substrate may be a combination of two or more othersubstrates, which, due to the combination, have become an identifiablenew substrate. In the embodiments of the invention, the substrate maycomprise metal, silicon, glass, or polymeric materials. In more specificembodiments, the substrate contains an integrated circuitry component,and is functionally coupled to a magnetic microcoil array, a vibrationelement, a detection element, and/or a circuit board.

A “microcoil” is a coil, or one or more connected loops, having at leastone dimension in the micrometer (μm), or less than 10⁻³ meter (mm),scale. A microcoil usually comprises a thin material wound or gatheredaround a center or an imaginative center into spiral, helical or othershapes. A microcoil is defined by the material itself, the shape of thewindings, and the separation between each windings. Solenoid typemicrocoils are multiple spiral wire loops, which may or may not bewrapped around a metallic core. A Solenoid type microcoil produces amagnetic field when an electrical current is passed through it and cancreate controlled magnetic fields. A Solenoid type microcoil can producea uniform magnetic field in a predetermined volume of space. A “planar”microcoil is a microcoil with its windings substantially remained in anactual or imaginative plane. Microcoils can also be fabricated into MEMSdevices such as demonstrated by MEMS magnetic actuators (IEEE Journal ofSolid-State Circuits (2006) 41:1471 and Biosensors and Bioelectronics(2006) 21:1693.

The embodiments of the invention contemplate the activation of one ormore microcoils (or to the movement of a permanent magnet) in order tomove the magnetic particles and/or binding complexes. “Activation” or“activating” refers to turning on one or more microcoils while turningoff (or keeping off) one or more other microcoils, which causes themagnetic particles (and any component attached to the magnetic particle)to move towards the microcoil(s) in the on position and away from themicrocoil(s) in the off position.

As used in the embodiments of the invention, “associated with” is usedinterchangeably with “functionally coupled” and means that two or moreobjects are so situated that the desired results or effects areachieved. For example, a microcoil array is “functionally coupled” withthe fluidic device when one or more microcoils are so situated that theywill achieve the desired effect of generating an magnetic field withinat least a portion of a fluidic zone of the device. Such coupling can bepermanent, where the microcoil array is integrated into the fluidicdevice, or temporary, where the microcoil array is adjacent or inproximity to the device but is not integrated into the device. Similarlya vibration element is also “functionally coupled” with the fluidicdevice when it is so situated that it will achieve the desired effect ofshaking, mixing, or agitating fluid within one or more fluidic zones ofthe device. Again, the vibration element can be integrated into thedevice, or can be in proximity to the device. In certain embodiments,the vibrational element agitates the fluid in one or more fluidic zonesto disperse the magnetic particles, analyte, and/or signal particles sothat they can interact to form a binding complex. In other embodiments,the vibrational element agitates the fluid in one or more fluidic zonesto facilitate aggregation-disaggregation and removal of unbound signalparticles and/or non-analyte components of the sample from the bindingcomplex. In another embodiment, the mixing is demonstrated on a MEMSdevices (IEEE Proc. Int. Conf. MEMS'02 (2002), 40-43). The detectionelement is “functionally coupled” with the fluidic device when it willachieve the desired effect of detecting and/or measuring the presence ofthe analyte (or binding complex) within the detection zone of thedevice. The detection element can be integrated into the device, or canbe in proximity to the device. The flow controller is “functionallycoupled” with the fluidic device when it will achieve the desired effectof coordinating liquid flow through the fluidic zones of the device.

A number of factors will be considered when associating the microcoilarray, the vibration element, the detection element, or the flowcontroller with the fluidic device, including the sizes and shapes ofthe substrate, the type and size of the microcoil array, the size andlocation of the fluidic zones, the number of the fluidic zones, thedesired strengths of the magnetic field and, and the volume within whichthe binding complex or signal particle is to be detected. As disclosedherein, the specific locations of the magnetic microcoil array, thedetection element and the vibration element with respect to the fluidicdevice will be determined based on the specific analysis desired by aperson skilled in the art.

As used herein, “dimension” or “dimensions” are the parameters ormeasurements required to define the shape and/or size, such as height,width, and length, of an object. As used herein, the dimension of atwo-dimensional object, such as a rectangle, a polygon, or a circle, isthe longest straight-line distance between any two points on the object.Therefore the dimension of a circle is its diameter; a rectangle itsdiagonal, and a polygon its longest diagonal. The dimension of athree-dimensional object is the longest straight-line distance betweenany two points on the object. The dimensions used herein are usuallymeasured by centimeters (cm), millimeters (mm), and micrometers (μm),and nanometers (nm).

A “fluidic device” or “fluidic network” is a device that has one or morefluidic zones that are capable of containing a liquid. A fluidic devicemay be functionally coupled to other components, such as a magneticmicrocoil array, a vibrational element, a detection element, a circuitboard and a circuitry component. Fluids used in the fluidic devicesinclude bodily fluids such as, but not limited to, amniotic fluid,aqueous humor, bile, blood and blood plasma, breast milk, cerebrospinalfluid, cerumen, colostrum, chyle, chyme, feces, female ejaculate,interstitial fluid, intracellular fluid, lymph, menses, mucus,pre-ejaculatory fluid, pleural fluid, pus, saliva, sebum, semen, serum,sweat, synovial fluid, tears, urine, vaginal lubrication, vitreoushumor, and vomit; bacterial cell suspensions; protein or antibodysolutions; various buffers; saline; and reaction substrates. The sampleintroduced into the fluidic device typically comprises a liquid, gel,solid, gas, or mixture thereof, suspected of containing an analyte.Fluidic devices can be used to obtain many interesting measurements,including fluid mechanical properties, cellular and molecular diffusioncoefficients, fluid viscosity, pH values, chemical and biologicalbinding coefficients and enzyme reaction kinetics. Other applicationsfor fluidic devices include cell and molecule detection and separation,capillary electrophoresis, isoelectric focusing, immunoassays, flowcytometry, sample injection of proteins for analysis via massspectrometry, DNA analysis, cell manipulation, and cell separation. Inone embodiment of the invention, magnetic materials and technologiesand/or nanoparticles are incorporated into the fluidic devices forapplications such as cell and biomolecule detection and/or separation.As used herein, the term “detecting the presence” refers to determiningthe existence, identity, and/or amount of an analyte in a particularsample.

A “fluidic zone” is typically a reservoir, channel, groove, opening, orconduit in the substrate of the fluidic device, which is configured forcontaining a liquid and optionally for containing reagents. Fluidiczones can be straight along their length, however, they can also containangles and curves of different degrees along their length. The fluidiczones can have rectangular cross-sections, or they may also have othershapes of cross-sections, such as circular. Typically the fluidic zonehas at least one dimension in the micrometer or millimeter scale.

The fluidic zones may be suitable for fluidic communications, such ascarrying a biological liquid to an adjacent fluidic zone. Alternatively,the fluidic zones may be suitable for non-fluidic communications, suchas carrying through molecules or compounds in the absence of significantactive hydraulic fluid transport or exchange. Such molecules orcompounds are typically magnetic particles, nanoparticles, affinityagents, analytes, and combinations thereof. The fluidic zones are oftenpart of an integrated device, such a fluidic device, such that liquid,molecules or compounds flowing through the fluidic zones occurs in acontrolled pattern and are able to be analyzed as desired.

The fluidic device typically comprises a plurality of fluidic zones. Inone embodiment, the plurality of fluidic zones comprises a sample zone,a cleaning zone and/or a detection zone. In a further embodiment, itcomprises more than one sample zone, cleaning zone, and/or detectionzone. It can comprise additional fluidic zones for storing reagents,which can be branches of any of the aforementioned zones. In oneembodiment, multiple fluidic zones are contained in parallel within thesame device, thus allowing for analysis of multiple samples or multipleanalytes in parallel. Each fluidic zone is separated from the adjacentfluidic zone by a diffusion barrier.

A “diffusion barrier” is a structure to minimize diffusion orconvectance of the contents of one fluidic zone to the next fluidiczone, such that the majority of the contents that move from one zone tothe next fluidic zone are moved by directed fluidic flow and/or byactivating the magnetic microcoil array. Diffusion barriers can becreated, for example, by elongating the channel, groove, opening orconduit (“the path of the fluidic zone”), narrowing the path, anglingthe path of the fluidic zone, or any combination thereof. Diffusionbarriers can also comprise a physical barrier, such asthermally-sensitive barrier. A “thermally-sensitive barrier” is aphysical barrier that becomes permeable due to the application of heat.For example, a thermally-sensitive barrier can comprise a gel that meltswhen heated and thus allows the contents of one fluidic zone to passthrough to the next zone. Hydrophilic fluid or liquid can be containedin a shape of droplets surrounded by hydrophobic liquid such as siliconeoils to form strong diffusion barriers through hydrophilic-hydrophobicinteractions so that droplets can be separated and transported withoutmixing with other fluids as demonstrated in J. Micromech. Microeng.(2006) 16:1875 and Sensors and Actuators B (2006) 113:563. A diffusionbarrier can be accomplished by “particle trapping and transport” throughDEP (dielectrophoresis) as demonstrated in Biophysical Journal (1998)74:1024 and Sensors and Actuators A 121 (2005) 59. In yet anotheraspect, the diffusion barrier can be created by a MEMS membrane valve.

The sample zone comprises a space for holding a sample, and is selectedfrom a reservoir, a channel, an opening, a surface, or a combinationthereof. In one embodiment, there is an inlet for allowing the insertionof a sample into the zone, and a vent to allow air or gas to exit as thesample is introduced. In a further embodiment, the vibration element isactivated to vibrate the fluid within the sample zone and deaggregatethe magnetic particles, signal particles, analyte, and/or bindingcomplexes, in order to facilitate interaction between these componentsand allow for separation of unbound components from the bindingcomplexes.

The cleaning zone is a reservoir, channel, groove, opening, or conduitconnecting the sample zone and the detection zone, which is preferablyseparated from the sample zone and detection zone by diffusion barriers.In one embodiment, an additional reaction between an analyte and amagnetic particle and/or a signal particle can occur in this zone. Inother embodiments, this zone provides a region whereby the magneticparticles and binding complexes are separated from unbound analyte orother components of the sample, and/or unbound signal particles. In afurther embodiment, the cleaning zone can comprise an affinity surfacethat is typically complementary to the affinity agent attached to themagnetic and/or signal particle, such that the particles are essentiallyimmobilized in this zone.

The detection zone is a reservoir, channel, groove, opening, or conduitin association with a detection element. It may comprise an array ofcapture molecules, as described in more detail below. The detectionelement can be an optical detection element or an electrical detectionelement. In certain embodiments, the optical detection element isselected from a Raman detector, a photon multiplier tube, a fluorescentreader, or an electrochemical sensor and the electrical detectionelement is selected from a FET element, a capacity detection element, acurrent sensor, and a charge sensor. Typically, the detection of thebinding complex or the signal analyte complex indicates the presence ofthe analyte.

In further embodiments, the detection zone comprises a reactionsubstrate. A “reaction substrate” is a material or substance upon whichan enzyme (such as the catalytic element) acts. The product of thereaction can be fluorogenic, chemiluminescent, or detectable byUV-visible light (such as by a color change). Non-limiting examples ofreaction substrates include Lumigen APS-5, Lumigen TMA-6, LumigenPS-atto, Lumigen PS-3, H₂O₂ with an oxidizable compound, Amplex Red,3,5,3′,5′-tetramethylbenzidine (TMB), glucose, O₂, ATP, Mg²⁺, luciferin,inoluciferin, quinolinyl, coelentrazine, aldehyde, FMNH₂, and analogsand combinations thereof.

Typically, if the detection zone comprises a reaction substrate, themagnetic affinity complex and/or the signal affinity complex comprises acatalytic element. The “catalytic element” is an external compound thatserves as an agent to cause a chemical reaction to occur in the reactionsubstrate, which reaction product is detectable by the detectionelement. In certain embodiments, the catalytic element is selected fromthe group consisting of alkaline phosphatase, horseradish peroxidase,glucose oxidase, luciferase (from firefly, Renilla, bacteria, or othersources) or analogs or combinations thereof.

The catalytic element can be conjugated to the signal particle through afunctionalized polymer. For example, a polymer with a functional group(i.e. aldehyde, amine, carboxylic acid, biotin) is used to conjugate theaffinity agent and/or catalytic element to the particle. Conjugation canbe through non-covalent interactions such as hydrophobic orelectrostatic interactions, or through covalent interactions, such asamide bond formation.

Additionally, the fluidic zones of the device can contain an appropriatebuffer to permit the reaction to occur.

Examples of non-limiting catalytic element-reaction substratecombinations and the method for detecting the reaction product are shownin Table 1.

TABLE 1 Examples Catalytic element Substrate Signal Detection 1 Alkalinephosphatase Lumigen APS-5 and others Light (450 nm) Photo sensor (AP) 2Horse-radish Lumigen PS-atto, Lumigen Light Photo sensor peroxidase(HRP) TMA-6, Lumingen PS-3, etc H₂O₂, oxidizable compound ElectronElectrical sensor Amplex Red (10-acetyl-3,7- Fluorescence fromexcitation at 530-571 nm, dihydroxyphenoxazine) + resorufin emission at590-600 nm H₂O₂ 3,5,3′,5′-tetramethylbenzidine Absorption (450 nm)UV-Vis (or its analogues) + H2O2 3 Glucose oxidase Glucose, O₂ ElectronElectrical sensor Glucose oxidase a) Glucose, O₂ for GO Fluorescencefrom excitation at 530-571 nm, (GO) + Horseradish b) Amplex Red for HRPresorufin emission at 590-600 nm peroxiadase (HRP) 4 Luciferase(firefly) ATP + MG2+ + O2 + luciferin light (560 nm) Photo sensor (orits analogues: aminoluciferin, quinolinyl luciferin) Luciferase(Renilla) Coelentrazine + O2 light (475 nm) Photo sensor Luciferase(Bacterial) Aldehyde + FMNH2 + O2 light (490 nm) Photo sensor

In certain embodiments, a fluorescent tag is attached to the signalparticle. Non-limiting examples of suitable fluorescent tags includeHcRed, green fluorescent protein, modified or enhanced green fluorescentprotein, yellow fluorescent protein, enhanced yellow fluorescentprotein, cyan fluorescent protein, blue fluorescent protein, redfluorescent protein, soluble-modified red-shifted green fluorescentprotein, soluble-modified blue fluorescent protein; blue variant ofgreen fluorescent protein; soluble-modified blue fluorescent protein, oranalogs or combinations thereof.

In other embodiments, the signal particle is itself detectable by thedetection element in the absence of a catalytic element and reactionsubstrate. Typically, in such situation, the signal particle willcomprise a SERS-active nanoparticle or a fluorescent nanoparticle, whichcan, for example, comprise a nanoparticle coupled to a surface-enhancedfluorescent tag. The SERS-active nanoparticle is detectable by Raman inthe detection zone. The fluorescent nanoparticle can be, for example, aQdot or other fluorescent nanoparticles, such as SEF nanoparticles orFluoDots, which are detectable by examining fluorescence in thedetection zone.

Alternatively, detection of the analyte can occur by fluorescencequenching. In one embodiment, the signal particle comprises ananoparticle coupled to an affinity agent and an ODN sequence. Thedetection zone contains a FRET pair of double stranded ODNs that containdonor or acceptor on one strand each, and where one of the singlestrands is complementary to the ODN sequence on the nanoparticle.Interaction between the ODN and the FRET pair results in a decrease influorescence, thus indicating the presence of the analyte.

In a further embodiment, the analyte is detected by FluorescenceResonance Energy Transfer (FRET). FRET is an energy transfer mechanismbetween two fluorescent molecules. A fluorescent donor is excited at itsspecific fluorescence excitation wavelength. By a long-rangedipole-dipole coupling mechanism, this excited state is thennonradiatively transferred to a second molecule, the acceptor, where itis then released as a photon. In one embodiment, a sandwich bindingcomplex is formed between a magnetic particle, an analyte, and ananoparticle coated with one partner of a FRET pair in one zone, thesandwich binding complex is moved to a second zone where another partnerof the FRET pair is present, and FRET is detected.

In a further embodiment, fluorescence is detected through the use ofcomplimentary segments of the fluorophore. For example, a sandwichbinding complex is formed between a magnetic particle, an analyte, and ananoparticle coated with half of a fluorescent protein. (such as GFP oran analog) in one zone, the sandwich binding complex is moved to asecond zone where the other half of the fluorescent protein is present,the fluorescent protein self-assembles, and fluorescence is detected.

Fluorogenic detection can also be employed. In one embodiment, a bindingcomplex is formed between a magnetic particle, an analyte, and anantibody-enzyme fusion protein in one zone, and it is moved to a secondzone containing a fluorogenic reaction substrate, where the enzymereacts with the reaction substrate to produce a detectable reactionproduct.

Time-resolved fluorescence can similarly be used in the invention. Inone embodiment, a sandwich binding complex is formed between a magneticparticle, an analyte, and a nanoparticle encoded with Eu³⁺ or Tb³⁺ oranother lanthanide in one zone; the binding complex is moved to anotherzone and detected by time-resolved fluorescence.

Other types of fluorescence can also be used, such as fluorescencepolarization, and fluorescence life time studies.

Additionally, binding complex formation can be detected bychemiluminescence. As described above, a sandwich binding complex can beformed between a magnetic particle, an analyte, and a nanoparticlecoated with a catalytic element in one zone, with chemiluminescentdetection in another fluidic zone. Alternatively, a binding complex canbe formed between a magnetic particle, an analyte, and anantibody-catalytic element fusion protein in one zone, withchemiluminescent detection in another zone. In a further embodiment, abinding complex can be formed between a magnetic particle, an analyte,and a silver nanoparticle or nanorod coated with affinity agents (suchas antibodies) for recognition of the analyte and chelating agents forits metal ions (such as Eu³⁺) in one zone, moving the binding complex toanother zone where metal ions such as Eu³⁺ are present, and detectingthe binding complex by chemiluminescence.

Binding complex formation is also detectable via UV-visiblespectroscopy. For example, a binding complex can be formed between amagnetic particle, an analyte and an antibody-catalytic element (such ashorseradish peroxidase) in one zone and the complex is moved to anotherzone where it reacts with one or more HRP substrates and is detected byUV-visible spectroscopy.

Reflectance can be used to detect binding complex formation. Forexample, a binding complex can be formed between a magnetic particle, ananalyte and a silver nanoparticle or nanorod coated with affinity agentspecific for the analyte in one zone, the binding complex is moved toanother zone, where it is detected by reflectance.

Binding complex formation can also be detected electrically, such as bycurrent measurement (where there is oxidation and reduction or freeelectron production), FET or potential measurement (where there is netor local charge changes), or by CHEM-FET, surface plasmon resonance,mass spectroscopy, interferometry, or radioactivity. These methods ofdetection are merely non-limiting examples of the many possible methodsof detecting the presence of a binding complex or signal particle in thedetection zone of the fluidic device of the invention.

The use of fluidic devices to conduct biomedical assays has manysignificant advantages. First, because the volume of fluids within thefluidic zones is very small, usually several nano-liters, the amount ofreagents and analytes required for the assays is quite small. This isespecially significant for expensive reagents. The fabricationstechniques used to construct these fluidic devices, discussed in moredetails herein, are relatively inexpensive and are very amenable both tohighly elaborated, multiplexed devices and also to mass production, suchas in an integrated circuit die. In manners similar to that formicroelectronics, fluidic technologies also enable the fabrication ofhighly integrated devices for performing different functions on the samesubstrate chip. Embodiments of the invention helps create integrated,portable clinical diagnostic devices for home and bedside use, therebyeliminating time consuming laboratory analysis procedures. Additionally,certain embodiments of the invention are self-contained such that liquiddoes not flow through the fluidic zones, thereby eliminating the needfor flow controllers. A self-contained fluidic network can also comprisepre-stored reagents, meaning during a test, no addition reagents need tobe added except for the sample, and water or buffer. In suchembodiments, the magnetic particles and any molecules bound to themagnetic particles are moved through the liquid contained within thefluidic zones by activating the magnetic microcoils, and are not movedby the flow of the liquid. Typically in these embodiments, the fluid ispresent in the fluidic zones to act as a suspending agent. Otherembodiments of the invention comprise a flow controller for coordinatingliquid flow through the fluidic zones of the device. In suchembodiments, the magnetic particles and any molecules bound to themagnetic particles are moved through the fluidic zones by activating themagnetic microcoils and/or also can be moved by activating the flowcontroller to move the liquid itself.

As used herein, “magnetic,” “magnetic effect,” and “magnetism” refer tothe phenomena by which one material exert an attractive or repulsiveforce on another material. Although theoretically all materials areinfluenced to one degree or another by magnetic effect, those skilled inthe art understand that magnetic effect or magnetism is only recognizedfor its detectability under the specific circumstance.

As used herein, a “permanent magnet” is a material that has a magneticfield without relying upon outside influences. Due to their unpairedelectron spins, some metals are magnetic when found in their naturalstates, as ores. These include iron ore (magnetite or lodestone),cobalt, and nickel. A “paramagnetic material” refers to a material thatattracts and repels like normal magnets when subject to a magneticfield. Paramagnetic materials include aluminum, barium, platinum, andmagnesium. A “ferromagnetic material” is a material that can exhibit aspontaneous magnetization. Ferromagnetism is one of the strongest formsof magnetism and is the basis for all permanent magnets. Ferromagneticmaterials include iron, nickel, and cobalt. A “superparamagneticmaterial” is a magnetic material that exhibits a behavior similar tothat of a paramagnetic material at temperatures below the Curie or theNeel temperature.

An “electromagnet” is a type of magnet in which the magnetic field isproduced by a flow of electric current. The magnetic field disappearswhen the current ceases. A simple type of electromagnet is a coiledpiece of wire that is electrically connected. An advantage of anelectromagnet is that the magnetic field can be rapidly manipulated overa wide range by controlling the electric current. In the embodiments ofthe invention, ferromagnetic or non-magnetic materials are used to formthe electromagnets.

An “array,” “macroarray” or “microarray” is an intentionally createdcollection of substances, such as molecules, openings, microcoils,detectors and/or sensors, attached to or fabricated on a substrate orsolid surface, such as glass, plastic, silicon chip or other materialforming an array. The arrays can be used to measure the expressionlevels of large numbers, e.g., tens, thousands or millions, of reactionsor combinations simultaneously. An array may also contain a small numberof substances, e.g., one, a few or a dozen. The substances in the arraycan be identical or different from each other. The array can assume avariety of formats, e.g., libraries of soluble molecules; libraries ofcompounds tethered to resin beads, silica chips, or other solidsupports. The array could either be a macroarray or a microarray,depending on the size of the pads on the array. A macroarray generallycontains pad sizes of about 300 microns or larger and can be easilyimaged by gel and blot scanners. A microarray would generally containpad sizes of less than 300 microns.

An array of magnetic microcoils is a collection of microcoils fabricatedon a substrate, such as silicon, glass, or polymeric substrate. Each ofthe microcoils may be associated with or functionally coupled to thefluidic device containing fluidic zones, across which the microcoil iscapable of generating a magnetic field as part of a biomedical assay.The fluidic zones may be a space for holding a liquid sample and/or asurface for immobilizing certain molecules, such as DNAs and proteins.The microcoil arrays may be a microarray or a macroarray depending onthe sizes or the microcoils and the associated sample spaces. In oneembodiment, the microcoil array is programmably activatable such thatindividual members or groups of the array turn on and off in acoordinated manner in order to move the magnetic particles (and anycompounds or molecules attached to the magnetic particles) from onefluidic zone to another fluidic zone. As used herein, “move” refers tochanging the position of the magnetic particle, and includesconcentrating and dispersing the particles as well as re-locating theparticles within a fluidic zone and/or from one fluidic zone to anotherfluidic zone.

A DNA microarray is a collection of microscopic DNA spots attached to asolid surface forming an array. DNA microarrays can be used to measurethe expression levels of large numbers of genes simultaneously. In a DNAmicroarray, the affixed DNA segments are known as probes, thousands ofwhich can be used in a single DNA microarray. Measuring gene expressionusing microarrays is relevant to many areas of biology and medicine,such as studying treatments, disease and developmental stages.

“Solid support” and “support” refer to a material or group of materialshaving a rigid or semi-rigid surface or surfaces. In some aspects, atleast one surface of the solid support will be substantially flat,although in some aspects it may be desirable to physically separatesynthesis regions for different molecules with, for example, wells,raised regions, pins, etched trenches, or the like. In certain aspects,the solid support(s) will take the form of beads, resins, gels,microspheres, or other geometric configurations.

The term “molecule” generally refers to a macromolecule or polymer asdescribed herein. However, channels or arrays comprising singlemolecules, as opposed to macromolecules or polymers, are also within thescope of the embodiments of the invention.

A “macromolecule” or “polymer” comprises two or more monomers covalentlyjoined. The monomers may be joined one at a time or in strings ofmultiple monomers, ordinarily known as “oligomers.” Thus, for example,one monomer and a string of five monomers may be joined to form amacromolecule or polymer of six monomers. Similarly, a string of fiftymonomers may be joined with a string of hundred monomers to form amacromolecule or polymer of one hundred and fifty monomers. The termpolymer as used herein includes, for example, both linear and cyclicpolymers of nucleic acids, polynucleotides, polysaccharides,oligosaccharides, proteins, polypeptides, peptides, phospholipids andpeptide nucleic acids (PNAs). The peptides include those peptides havingeither α-, β-, or ω-amino acids. In addition, polymers includeheteropolymers in which a known drug is covalently bound to any of theabove, polyurethanes, polyesters, polycarbonates, polyureas, polyamides,polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides,polyacetates, or other polymers which will be apparent upon review ofthis disclosure.

The term “biomolecule” refers to any organic molecule that is part of orfrom a living organism. Biomolecules include a nucleotide, apolynucleotide, an oligonucleotide, a peptide, a protein, a ligand, anantibody, a receptor, among others. A “complex of a biomolecule” refersto a structure made up of two or more types of biomolecules. Examples ofa complex of biomolecule include a cell or viral particles.

As used herein, “biological cells” and “cells” are interchangeable,unless otherwise clearly indicated, and refer to the structural andfunctional units of all living organisms, sometimes called the “buildingblocks of life.” Cells, as used herein include bacteria, fungi, andanimal mammalian cells. Specifically included are animal blood cells,such as red blood cells, white blood cells, and platelets.

The term “analyte” or “analyte molecule” refers to a molecule orbiological cell of interest that is to be analyzed or detected, e.g., anucleotide, an oligonucleotide, a polynucleotide, a peptide, a protein,an antibody, or a blood cell. Examples of analytes that can beinvestigated by this invention include, but are not restricted to,agonists and antagonists for cell membrane receptors, toxins and venoms,viral epitopes, hormones, hormone receptors, peptides, enzymes, enzymereaction substrates, cofactors, drugs (e.g. opiates, steroids, etc.),lectins, sugars, polynucleotides, nucleic acids, oligosaccharides,proteins, antibodies, and autoantibodies. The analyte or analytemolecule could be a small molecule, biomolecule, or nanomaterial such asbut not necessarily limited to a small molecule that is biologicallyactive, nucleic acids and their sequences, peptides and polypeptides, aswell as nanostructure materials chemically modified with biomolecules orsmall molecules capable of binding to molecular probes such aschemically modified carbon nanotubes, carbon nanotube bundles, nanowiresand nanoparticles. The analyte in an assay can be a moiety or derivativegenerated by assay process, which is the subsequently recognized anddetected as surrogate marker of the analyte contained in the sample. Theanalyte may be magnetically tagged, or labeled to facilitate itsdetection and separation.

The term “affinity agent” refers to a molecule that binds to an analytefor the detection and/or analysis of the analyte. The affinity agentgenerally, but not necessarily, has a known molecular structure orsequence. In one embodiment, the affinity agent is attached to a solidsurface of the fluidic device. When the affinity agent is attached to asolid surface, it is referred to as an “affinity surface”. In anotherembodiment, the affinity agent is attached to a magnetic particle orsignal particle. When the affinity agent is attached to the magneticparticle, it is referred to as a “magnetic affinity complex”. When theaffinity agent is attached to the signal particle, it is referred to asa “signal affinity complex”. In one embodiment of the signal affinitycomplex, the affinity agent is the analyte of interest; in such case,the signal affinity complex is termed a “signal analyte complex”. Theaffinity agent typically include, but are not limited to antibodies,autoantibodies, cell membrane receptors, monoclonal or polyclonalantibodies and antisera reactive with specific antigenic determinants(such as on viruses, cells or other materials), drugs, polynucleotides,nucleic acids, peptides, proteins, cofactors, lectins, sugars,polysaccharides, cells, cellular membranes, and organelles. Affinityagents are biomolecules capable of undergoing binding or molecularrecognition events with analytes. An affinity agent can be a capturemolecule.

The term “capture molecule” refers to a molecule that is immobilized ona surface. The capture molecule can bind to the analyte, the magneticparticle, the signal particle, the affinity agent, or the code. Thecapture molecule is typically a nucleotide, an oligonucleotide, apolynucleotide, a peptide, or a protein, but could also be a smallmolecule, biomolecule, or nanomaterial such as but not necessarilylimited to a small molecule that is biologically active, nucleic acidsand their sequences, peptides and polypeptides, as well as nanostructurematerials chemically modified with biomolecules or small moleculescapable of binding to an analyte that is bound to an affinity agent toform a complex of the capture molecule, analyte and the magneticaffinity complex and/or the signal affinity complex. The capturemolecule may be magnetically or fluorescently labeled DNA or RNA. Inspecific embodiments of the invention, the capture molecule may beimmobilized on the surface of a fluidic zone of the fluidic device. Thecapture molecule may or may not be capable of binding to just theanalyte, or just the affinity agent.

The terms “die,” “polymer array chip,” “DNA array,” “array chip,” “DNAarray chip,” or “bio-chip” are used interchangeably and refer to acollection of a large number of probes arranged on a shared substratewhich could be a portion of a silicon wafer, a nylon strip or a glassslide.

Certain embodiments of the invention contemplate the use of codedmagnetic particles and signal particles for detecting the presence of ananalyte using the devices described herein. Typically, a samplesuspected of comprising an analyte is introduced into the sample zone ofthe fluidic device, wherein a coded magnetic affinity complex binds tothe analyte to form a coded magnetic binding complex. The microcoilarray is activated to move the coded magnetic binding complex from thesample zone to a first affinity surface, where it is bound andimmobilized. Typically the affinity agent on the first affinity surfaceis complementary to and binds to the affinity agent on the magneticparticle. The code is then detached from the coded magnetic bindingcomplex. The detached code then binds to a magnetic signal affinitycomplex to form a coded magnetic signal binding complex. Typically theaffinity agent of the magnetic signal affinity complex is complementaryto the code. In one embodiment, the affinity agent of the magneticsignal affinity complex is a polynucleotide complementary to the codepolynucleotide. The microcoil array is activated to move the codedmagnetic signal binding complex to one or multiple detection zonescomprising a second affinity surface. Typically different areas of thedetection zone or the different detection zones contain unique affinityagents to the codes. The affinity agents of the second affinity surfaceare complementary to and bind the code. The detection element thendetects the coded magnetic signal binding complex in the detection zoneusing electrical sensing methods, optical sensing methods, or enzymaticmethods, such as amplifying the affinity agent (if it is apolynucleotide) on the magnetic signal affinity complex.

“Detach” refers to the separation of the code molecule from the affinityagent of the magnetic particle. It can be detached using any methodknown to those of skill in the art. In one embodiment, it is detached byheating. In other embodiments, it is enzymatically detached.

The term “nucleotide” includes deoxynucleotides, ribonucleotides andanalogs thereof. These analogs are those molecules having somestructural features in common with a naturally occurring nucleotide suchthat when incorporated into a polynucleotide sequence, they allowhybridization with a complementary polynucleotide in solution.Typically, these analogs are derived from naturally occurringnucleotides by replacing and/or modifying the base, the ribose or thephosphodiester moiety. The changes can be tailor-made to stabilize ordestabilize hybrid formation, or to enhance the specificity ofhybridization with a complementary polynucleotide sequence as desired,or to enhance stability of the polynucleotide.

The term “polynucleotide” or “nucleic acid” as used herein refers to apolymeric form of nucleotides of any length, either ribonucleotides ordeoxyribonucleotides, that comprise purine and pyrimidine bases, orother natural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. Polynucleotides of the embodiments of theinvention include sequences of deoxyribopolynucleotide (DNA),ribopolynucleotide (RNA), or DNA copies of ribopolynucleotide (cDNA)which may be isolated from natural sources, recombinantly produced, orartificially synthesized. A further example of a polynucleotide of theembodiments of the invention may be polyamide polynucleotide (PNA) orlinked polynucleotide (LNA). The polynucleotides and nucleic acids mayexist as single-stranded or double-stranded. The backbone of thepolynucleotide can comprise sugars and phosphate groups, as maytypically be found in RNA or DNA, or modified or substituted sugar orphosphate groups. A polynucleotide may comprise modified nucleotides,such as methylated nucleotides and nucleotide analogs. The sequence ofnucleotides may be interrupted by non-nucleotide components. Thepolymers made of nucleotides such as nucleic acids, polynucleotides andpolynucleotides may also be referred to herein as “nucleotide polymers.

When the biomolecule or macromolecule of interest is a peptide, theamino acids can be any amino acids, including α, β, or ω-amino acids.When the amino acids are α-amino acids, either the L-optical isomer orthe D-optical isomer may be used. Additionally, unnatural amino acids,for example, β-alanine, phenylglycine and homoarginine are alsocontemplated by the embodiments of the invention. These amino acids arewell-known in the art.

A “peptide” is a polymer in which the monomers are amino acids and whichare joined together through amide bonds and alternatively referred to asa polypeptide. In the context of this specification it should beappreciated that the amino acids may be the L-optical isomer or theD-optical isomer. Peptides are two or more amino acid monomers long, andoften more than 20 amino acid monomers long.

A “protein” is a long polymer of amino acids linked via peptide bondsand which may be composed of two or more polypeptide chains. Morespecifically, the term “protein” refers to a molecule composed of one ormore chains of amino acids in a specific order; for example, the orderas determined by the base sequence of nucleotides in the gene coding forthe protein. Proteins are essential for the structure, function, andregulation of the body's cells, tissues, and organs, and each proteinhas unique functions. Examples are hormones, enzymes, and antibodies.

The term “sequence” refers to the particular ordering of monomers withina macromolecule and it may be referred to herein as the sequence of themacromolecule.

The term “hybridization” refers to the process in which twosingle-stranded polynucleotides bind non-covalently to form a stabledouble-stranded polynucleotide; triple-stranded hybridization is alsotheoretically possible. The resulting (usually) double-strandedpolynucleotide is a “hybrid.” The proportion of the population ofpolynucleotides that forms stable hybrids is referred to herein as the“degree of hybridization.” For example, hybridization refers to theformation of hybrids between a probe polynucleotide (e.g., an affinityagent polynucleotide of the invention which may include substitutions,deletion, and/or additions) and a specific analyte polynucleotidewherein the probe preferentially hybridizes to the specific analytepolynucleotide and substantially does not hybridize to polynucleotidesconsisting of sequences which are not substantially complementary to theanalyte polynucleotide. However, it will be recognized by those of skillthat the minimum length of a polynucleotide desired for specifichybridization to a target polynucleotide will depend on several factors:G/C content, positioning of mismatched bases (if any), degree ofuniqueness of the sequence as compared to the population of analytepolynucleotides, and chemical nature of the polynucleotide (e.g.,methylphosphonate backbone, phosphorothiolate, etc.), among others.

Methods for conducting polynucleotide hybridization assays have beenwell developed in the art. Hybridization assay procedures and conditionswill vary depending on the application and are selected in accordancewith the general binding methods known in the art.

It is appreciated that the ability of two single strandedpolynucleotides to hybridize will depend upon factors such as theirdegree of complementarity as well as the stringency of the hybridizationreaction conditions.

As used herein, “stringency” refers to the conditions of a hybridizationreaction that influence the degree to which polynucleotides hybridize.Stringent conditions can be selected that allow polynucleotide duplexesto be distinguished based on their degree of mismatch. High stringencyis correlated with a lower probability for the formation of a duplexcontaining mismatched bases. Thus, the higher the stringency, thegreater the probability that two single-stranded polynucleotides,capable of forming a mismatched duplex, will remain single-stranded.Conversely, at lower stringency, the probability of formation of amismatched duplex is increased.

The appropriate stringency that will allow selection of aperfectly-matched duplex, compared to a duplex containing one or moremismatches (or that will allow selection of a particular mismatchedduplex compared to a duplex with a higher degree of mismatch) isgenerally determined empirically. Means for adjusting the stringency ofa hybridization reaction are well-known to those of skill in the art.

The term “chip” or “microchip” refers to a small device or substratethat comprises components for performing certain functions. A chipincludes substrates made from silicon, glass, metal, polymer, orcombinations and capable of functioning as a microarray, a macroarray, afluidic device, and/or an integrated circuitry component. A chip may bea microelectronic device made of semiconductor material and having oneor more integrated circuits or one or more devices. A “chip” or“microchip” is typically a section of a wafer and made by slicing thewafer. A “chip” or “microchip” may comprise many miniature transistorsand other electronic components on a single thin rectangle of silicon,sapphire, germanium, silicon nitride, silicon germanium, or of any othersemiconductor material. A microchip can contain dozens, hundreds, ormillions of electronic components. In the embodiments of the invention,as discussed herein, fluidic zones, magnetic microcoil arrays, detectionelements, and vibration elements can also be integrated into amicrochip.

“Micro-Electro-Mechanical Systems (MEMS)” is the integration ofmechanical elements, sensors, actuators, and electronics on a commonsilicon substrate through microfabrication technology. While theelectronics are fabricated using integrated circuit (IC) processsequences (e.g., CMOS, Bipolar, or BICMOS processes), themicromechanical components could be fabricated using compatible“micromachining” processes that selectively etch away parts of thesilicon wafer or add new structural layers to form the mechanical andelectromechanical devices. Microelectronic integrated circuits can bethought of as the “brains” of a system and MEMS augments thisdecision-making capability with “eyes” and “arms”, to allow microsystemsto sense and control the environment. Sensors gather information fromthe environment through measuring mechanical, thermal, biological,chemical, optical, and magnetic phenomena. The electronics then processthe information derived from the sensors and through some decisionmaking capability direct the actuators to respond by moving,positioning, regulating, pumping, and filtering, thereby controlling theenvironment for some desired outcome or purpose. Because MEMS devicesare manufactured using batch fabrication techniques similar to thoseused for integrated circuits, unprecedented levels of functionality,reliability, and sophistication can be placed on a small silicon chip ata relatively low cost. In the embodiments of the invention, as discussedherein, MEMS devices can be further integrated with fluidic zones,diffusion barriers, magnetic microcoil arrays, detection elements,and/or vibration elements, such that, together, they perform separationand detection function for biomolecules.

An “integrated circuitry component” is a processor on an integratedcircuit (IC) chip. The processor may be one or more processor on one ormore IC chip. The chip is typically a silicon chip with thousands ofelectronic components that serves as a central processing unit (CPU) ofa computer or a computing device. It is typically a readable andwritable memory chip, with or without contact. In certain embodiments,it can store reagent information, operation instructions and programs,and test results and data.

A “nanomaterial” as used herein refers to a structure, a device or asystem having a dimension at the atomic, molecular or macromolecularlevels, in the length scale of approximately 1-1000 nanometer (nm)range. Preferably, a nanomaterial has properties and functions becauseof the size and can be manipulated and controlled on the atomic level.

The term “complementary” refers to the topological compatibility ormatching together of interacting surfaces of an analyte and itscorresponding affinity agent. Thus, the affinity agent and its analytecan be described as complementary, and furthermore, the contact surfacecharacteristics are complementary to each other. With respect topolynucleotides, sequences are complementary when they are able tohybridize to each other to form a stabilized duplex.

One embodiment of the invention relates to a device for detecting thepresence or amount of an analyte in a sample. The device comprises afluidic network and an integrated circuitry component which isfunctionally coupled to a magnetic microcoil array, a detection element,a circuit board, and optionally, a vibration element. In the embodiment,the fluidic network comprises a plurality of fluidic zones, where eachzone is connected to the adjacent zone by a diffusion barrier. One ormore of the fluidic zones contains a magnetic particle and a signalparticle. A sample suspected of containing an analyte is introduced intoa fluidic zone. The analyte interacts with a magnetic particle and thesignal particle to form a binding complex. The magnetic microcoil arrayis to generate a magnetic field across at least a portion of a fluidiczone to move the binding complex to a fluidic zone where it can bedetected by the detection element.

In one embodiment, the fluidic device integrates fluidic zones and amicrocoil for generating a magnetic field within a portion of a fluidiczone. The fluidic zones and the microcoil may be supported by orintegrated into a substrate. In another embodiment, the microcoil isplaced near the substrate. When activated, the microcoil generates amagnetic field within a portion of a fluidic zone.

In a specific embodiment of the invention, the detection element of thesubstrate comprises silicon, glass, a polymeric material, metal, or acombination thereof. More specifically, the detection element may eithercomprise or be connected to an integrated circuit, a MEMS device, amicroarray, a macroarray, a fluidic device, or a combination thereof. Inother words, the embodiment can be integrated into or connected to awide range of materials used in a variety of existing devices.

Silicon is a suitable material for forming micro-channels coupled withmicroelectronics or other microelectromechanical systems (MEMS). It alsohas good stiffness, allowing the formation of fairly rigidmicrostructures, which can be useful for dimensional stability. In aspecific embodiment of the invention, the fluidic device or substratecomprises an integrated circuitry element (IC), a packaged integratedcircuit, and/or an integrated circuit die. For example, the substratemay be a packaged integrated circuit that comprises a microprocessor, anetwork processor, or other processing device. The substrate may beconstructed using, for example, a Controlled Collapse Chip Connection(or “C4”) assembly technique, wherein a plurality of leads, or bond padsare internally electrically connected by an array of connection elements(e.g., solder bumps, columns).

Specific materials useful as the substrate also include, but not limitedto, polystyrene, polydimethylsiloxane (PDMS), glass, chemicallyfunctionalized glass, polymer-coated glass, nitrocellulose coated glass,uncoated glass, quartz, natural hydrogel, synthetic hydrogel, plastics,metals, and ceramics. The substrate may comprise any platform or devicecurrently used for carrying out immunoassays, DNA or protein microarrayanalysis. Thus, the substrate may comprise a microarray or a macroarray,a multi-well plate, a fluidic device, or a combination thereof.

In another embodiment, the fluidic device comprises circuitry that iscapable of amplifying or processing the optical or electrical signalsdetected by the detection element. Any suitable conventional circuitsmay be used and integrated into the substrate for amplifying and/orprocessing, including filtering, the optical or electrical signalsdetected and collected by the detection element. The integratedcircuitry may be able to generate a read-out of the optical orelectrical signal independently or can be connected to an externaldevice for generating the read-out.

In another embodiment of the invention, the sample is a liquid, a gel, asolid, a gas, or a mixture thereof. Therefore, the embodiment of theinvention can accommodate samples in different physical states. In aspecific embodiment, the sample is a liquid or in a liquid or solutionstate. In another embodiment, the sample zone comprises a reservoir, achannel, an opening, a surface, or a combination thereof. The embodimentaccommodates a variety of applications in which a sample suspected ofcontaining an analyte is to be analyzed. For example, the sample zonemay be a reservoir, an opening void, or a surface that can hold a liquidsample. In such cases, the sample zone may be an open reservoir orsurface, or a substantially closed void with an opening for sampleinput. The design of the space depends not only on the specific analysisto be done, but also on how to best situate and design the sampleholding space in relation to the associated microcoil, detectionelement, and vibration element, as discussed herein.

According another embodiment, the sample zone for holding a sample, suchas a liquid sample, may also be the whole or part of a channelfabricated on the substrate. Depending on the specific requirement, thechannel may be open (a trench) or closed. The channel typicallycomprises an inlet and an outlet, but may also comprise other openingsfor fluidic communication. In another embodiment, the channel comprisestwo or more inlets and at least one outlet such that different reactantsmay be introduced into the channel from different inlets and mixed at amixing section within the channel for specific chemical reaction.Furthermore, the channel may comprise more than two inlets and more thanone mixing section such that more than one reaction may occur withindifferent sections of the channel according predetermined manners. Asdiscussed herein, the channel is designed in consideration with itsrelations with the associated microcoil, detection element, andvibration element to achieve the desired optical or electrical signal todetect the presence of the analyte.

In the embodiments of the invention, the sample zone of the device canaccommodate a wide range of sample volume, including very small amountof samples. In one embodiment, the sample zone has a volume of fromabout 1.0 mL to about 1.0 mL. In another embodiment, the sample zone hasa volume of from about 10 nL to about 10 μL. As understood by a personskilled in the art, actual sample volumes will depend on the nature ofthe analysis to be conducted, in addition to the design and dimensionsof the device. In cases where the sample zone is a channel having twoinlets and one outlet, the total sample zone may be substantially largerthan the volume that is in proximity to a particular microcoil. Forexample, the total channel volume, excluding the inlets and outlet, maybe about 1.0 μm while the volume in proximity to the microcoil may beabout only 10 nL to 100 nL.

In the embodiments of the invention, many conductive materials aresuitable for the microcoils. In the embodiments, the microcoil can beused for generating an excitation magnetic field across at least aportion of a fluidic zone. The selection of materials for the microcoildepends on several factors including the type and size of the coil, thedesired strength of the magnetic field, the size and location of eachfluidic zone, the shape, size and nature of the substrate, and thelocations of the vibration element and detection element. Theconductivity of the material is important to the selection. In oneembodiment of the invention, the microcoil comprises copper, aluminum,gold, silver, or a mixture thereof.

In the embodiments of the invention, the microcoil is “functionallycoupled” with the fluidic zones. A number of factors will be consideredwhen functionally coupling the microcoil with the space, including thetype and size of the microcoil, the sizes and locations of each fluidiczone, the desired strength of the magnetic field, and the volume withinwhich the magnetic field will be effectuated. In a specific embodiment,the microcoil is placed near or adjacent to the fluidic zone. Thespecific type, size, strength, and location of the microcoil on thesubstrate will be determined based on the specific analysis desired by aperson skilled in the art.

In one embodiment of the invention, the microcoil is a Solenoid typecoil. Solenoid type microcoils are multiple spiral wire loops, which mayor may not be wrapped around a metallic core. A Solenoid type microcoil,in addition to serving as a detection circuit, produces a magnetic fieldwhen an electrical current is passed through it and can createcontrolled magnetic fields. In the embodiment of the invention, theSolenoid type microcoil can produce a uniform magnetic field in apredetermined volume of the fluidic zone.

According to another embodiment of the invention, existing technologiescan be used to construct the devices of the invention. For example,silicon process technologies can be used to construct or fabricate thefluidic device of the embodiments of the invention, such that thefluidic zones, diffusion barriers, and optionally the microcoils andvibration element can be constructed on a substrate that may alsocomprise an integrated circuitry component and/or microfluidicmechanisms such as flow controllers. In another embodiment,servo-mechanical components and mechanisms can be used to control thelocation and movement of the detection element such that the desiredsignals are detected.

FIGS. 1-14 illustrate various embodiments of the invention.

FIG. 1 illustrates an embodiment of the invention that comprises afluidic network in association with a magnetic microcoil array, adetection element, an integrated circuitry component, and is in furtherassociation with a circuit board. As illustrated, the fluidic networkcontains a sample zone, a detection zone, and another fluidic zonebetween the sample zone and the detection zone. The sample is loadedinto the sample zone, where analyte present in the sample forms acomplex with a magnetic particle. The complex is moved through thefluidic zones to the detection zone by activating the microcoil array.It is then detected in the detection zone by the detection element. Thedevice is further connected with a circuitry component and circuitboard, which collects, analyzes, and/or processes signals detected bythe detection element.

FIG. 2 illustrates magnetic particles overlaying an exemplary magneticmicrocoil array, demonstrating the movement of magnetic particles (darkcircles) over the magnetic coils. As shown, the microcoils are activated(turned on and off in a directed fashion) to move the magnetic particlesfrom left to right. Molecules that are coupled to the magnetic particlesare also moved by activating the microcoils.

FIG. 3 illustrates a top-down view and cross-section view of the fluidicnetwork. The cross-section view illustrates the functionally coupledmagnetic microcoil array. As shown, the sample is introduced into thesample zone. There are optional fluidic zones for storing reagents,which contain one or more sets of magnetic particles. The underlyingmagnetic array (which can be integrated or in a separate, coupleddevice) is activated to move the magnetic particles into the samplezone. In another embodiment (not shown), the magnetic particles arepresent within the sample zone, and are not located in the storageareas. There is also a waste zone: magnetic particles can be moved intothe waste zone and uncomplexed analyte can be left in this area. Themagnetic array is activated to move the magnetic particles and complexedanalyte into the detection zone. The detection zone can contain one ormore different regions (indicated by 1-4) for detection of differentanalytes.

FIG. 4 illustrates a more detailed top-down view and cross-section viewof the fluidic network functionally coupled to the magnetic microcoilarray, providing examples of various diffusion barriers. As shown, themicrocoil array is indicated by the dashed ovals in the top-down view,and by the squares in the cross-section view. The sample is insertedinto the sample zone through a loading inlet, where it interacts withmagnetic particles and the analyte binds to the magnetic particle. Themagnetic particles are optionally moved into the branch, which is afluidic zone containing one or more reagents. The magnetic microcoilarray is activated to move the magnetic particles (complexed anduncomplexed) through a diffusion barrier to the cleaning zone. Themagnetic particles are further moved into the detection zone, fordetection by the detection element. The integrated circuitry componentsaves data in its memory.

FIG. 5 illustrates a cross-section view of the fluidic network andexemplary method for detecting an analyte. The fluidic network isfunctionally coupled to the magnetic microcoil array, and contains amagnetic affinity complex, an analyte and a signal affinity complex. Themagnetic affinity complex (“M”) interacts with the analyte and thesignal affinity complex to form a sandwich binding complex. Thevibration element is optionally employed to deaggregate the magnetic andsignal particles and the analyte, and to allow them to interact. Themicrocoil array is activated to move the magnetic affinity complexes tothe detection zone. Unbound signal affinity complex is not moved to thedetection zone. While both complexed and uncomplexed magnetic particlesare in the detection zone, the signal is generated only by the signalparticles that have interacted with the analyte and the magneticaffinity complex. Both optical and electrical signals can be detected.The signal indicates the presence of the analyte. FIG. 6A illustratesthe use of the fluidic network such as a biochip. Magnetic particles andQdots were loaded into the fluidic network. The magnetic microcoils wereactivated and the magnetic particles (indicated by the arrows) movedfrom the sample zone in panel 1 through the fluidic channels and intothe detection zone by panel 6. Note that in panel 3, the magneticmicrocoils were activated to spread out the magnetic particles. Thefluorescent images illustrate that the Qdots did not move from thesample zone. FIG. 6B illustrates the fluorescence of a mixture ofmagnetic particles and Qdots. As the mixture is washed, it losesfluorescence. FIG. 6C quantifies the fluorescence of the samples in thetubes from FIG. 6B (S1 tube is the original sample, while S2-S4 tubesare the washes) or samples taken from the fluidic network during FIG. 6A(“initial-on chip” indicates the sample zone after the magneticparticles have been moved, while “end-on chip” indicates the sample fromthe detection zone”). FIG. 6D illustrates on-chip chemiluminescentdetection of PSA. Comparative studies were carried out by dividingsamples into two-halves, one half for on-chip test and the other forin-tube test. The on-chip test demonstrated the capability of removingsignaling particles from sandwich complex by magnetic transport. FIG. 6Dquantifies chemiluminescence photo counts corresponding to the analyte,free PSA, for the “on-chip” experiment performed with fluidic network aswell as for the “in-tube” (multi-steps) experiment. The on-chip testshowed very comparative results to the in-tube test.

FIG. 7 illustrates the formation of sandwich binding complexes(“sandwich binding” and “tandem binding”) and a competitive bindingcomplex. A sandwich binding complex is formed through the analytebinding to a signal affinity complex and a magnetic affinity complex.The “sandwich binding” shows an example where the analyte is a proteinor nucleic acid, while the “tandem binding” shows an example where theanalyte is an antibody. An optical or electrical signal is detected fromthe binding complex. A competitive binding complex is formed when asignal analyte complex displaces the analyte from a magnetic bindingcomplex (analyte plus magnetic affinity complex). Signal is detectedfrom the signal analyte complex that does not form the competitivebinding complex.

FIG. 8 illustrates the use of codes with a magnetic affinity complex anda magnetic signal affinity complex. The coded magnetic affinity complexcomprises a code, an affinity agent and a magnetic particle. It caninteract with the analyte to form a coded magnetic binding complex. Themicrocoil array is activated to move the coded magnetic binding complexto a first affinity surface, where it is bound and immobilized. In thisexample, the affinity agent on the first affinity surface iscomplementary to and binds to the affinity agent on the magneticparticle. The code is then detached from the coded magnetic bindingcomplex. The detached code then binds to a magnetic signal affinitycomplex to form a coded magnetic signal binding complex. Typically theaffinity agent of the magnetic signal affinity complex is complementaryto the code. In this example, the affinity agent of the magnetic signalaffinity complex is a polynucleotide complementary to the codepolynucleotide. The microcoil array is activated to move the codedmagnetic signal binding complex to one or multiple detection zonescomprising a second affinity surface. Typically different areas of thedetection zone or the different detection zones contain unique affinityagents to the codes. The affinity agents of the second affinity surfaceare complementary to and bind the code. The detection element thendetects the coded magnetic signal binding complex in the detection zoneusing electrical sensing methods, optical sensing methods, or enzymaticmethods, such as amplifying the affinity agent (if it is apolynucleotide) on the magnetic signal affinity complex.

FIG. 9 illustrates an embodiment of the particle (or molecule) transportdevice of the invention, showing major components which are (1) afluidic network, e.g., a biochip, (2) an electromagnetic array, (3) acircuitry board; and (4) computer. The fluidic network comprises aplurality of fluidic zones, each fluidic zone being connected to theadjacent zone by a diffusion barrier, and an integrated circuitrycomponent, and optionally has a vibration element functionally coupledto the fluidic network. The array of magnetic microcoils functionally iscoupled to the fluidic network, wherein the microcoils are programmablyactivatable to generate a magnetic field in proximity to each microcoil.The electromagnetic array can concentrate or transport magneticparticles, but dispersion of magnetic particles is preferably done bythe vibrational device, which could be integrated in the fluidicnetwork. A detection element (not shown in FIG. 9) could be functionallycoupled to the fluidic network.

The circuitry board shown in FIG. 9 contains the circuitry to controlthe elements (core/coil) of the electromagnetic array. The circuitryboard is connected, either hard-wired or by wireless connection, to acomputer or any other device for controlling the switches of thecircuitry board in a preferred sequence. The computer or any processingunit could include an embedded computer processor and/or could becapable of integrated computing.

Particle transport in the fluidic device is achieved by using themagnetic array and magnetic particles. Magnetic particles arecommercially available. For clinical diagnostic applications, theparticles could be conjugated with affinity binding partners (e.g.nucleic acid probes or antibodies); they could also be used togetherwith other nanoparticles which can serve as either signal source or ascarriers of signal sources. Magnetic particles and other reagents areplaced in the fluidic device (e.g., biochip) containing multiple zoneswherein liquid transport is not needed, and thus mechanism to generatefluid movement force is avoided.

To facilitate biomolecule detection, aggregated or concentratedparticles in the fluidic zones may need to be dispersed, mixed, orresuspended in solution locally within in a fluidic zone. Dispersing canbe achieved by mechanical means, e.g., the vibrational device such asultrasounds (acoustic), piezo vibrations. The dispersing elements can befunctionally coupled to the nBMA device (integrated with chip or thecontrol device).

In one embodiment, the electromagnetic array comprises magnetic core,e.g., Fe cores, surrounded by power coils, preferably a set of planarcoils. As shown in FIG. 10, the cross sectional areas of the core of theelectromagnet could have various geometries such as a star (FIG. 10,bottom left) or a circle (FIG. 10, bottom right). Generally, coreshaving the star cross-section produce a more even magnetic filed betweentwo adjacent cores while cores having the circular shape produce aconcentrated magnetic field in the region where two adjacent cores areclosest. Thus, by appropriate choice of the cross-sectional areas of thecores, the electromagnetic array could have regions with substantiallyuniform or concentrated magnetic field.

The electromagnetic array creates magnetic field gradients that aresufficient to transport the magnetic particles in the fluidic device.The power coils can be switched by the switching circuitry, which inturn can be controlled by a computer. The switching could be on/off,high/low and/or at a desired frequency, which can be determined as afunction of the time necessary for a magnetic particle to be transportedwithin the fluidic device.

FIGS. 11A and B show an embodiment of the switching circuitry. Thecomputer generates low current signals which are used to control thehigh current needed for the electromagnets. The current for each coilcan be controlled by either a solid state or electromechanical relay orcurrent amplifier which is driven by a logical or analog signalgenerated by the computer control. FIG. 11A is an example of a circuitfor an individual magnetic element for which the polarity can beswitched. So one would need two switches per element of the magneticarray. However, by the switching circuitry of FIG. 11B, it would bepossible to minimize the number of switches but keep the polarity fixedsuch that, for example, for N elements containing N coils, one wouldneed just 25+N/5 switches.

FIG. 12 illustrates an example of the movement of magnetic particles ona microscope slide. Initially, in FIG. 12 (1), a liquid solutioncontaining colored magnetic particles was spread out on a portion of theslide overlaying directly above three core/coil elements. Note that themicroscope slide in FIG. 12 has been moved down to take the picture, butin the experiment, the liquid solution was on top of the three core/coilelements. Next, all three elements were switched on to have north (N)polarity. As a result, as illustrated in FIG. 12 (2), the magneticparticles in the liquid solution separated into two distinct regionsabove the first and third core/coil elements. Next, the three elementswere switched on to have south (S), N, N polarity. In this case, asillustrated in FIG. 12 (3), the majority of the magnetic particles movedto a spot between S and N polarity elements and some magnetic particlesformed a spot above the third element having N polarity. Next, the threeelements were switched to have zero (no), S, N polarity. In this case,as illustrated in FIG. 12 (4), the magnetic particles moved to a spotbetween the second and third elements having S and N polarity. Finally,the three elements were switched on to have zero, zero and N polarity.In this case, as illustrated in FIG. 12 (5), the magnetic particlesmoved to a spot above the third element having N polarity. This exampleclearly demonstrates that a magnetic array within the embodiments of theinvention can transport and/or concentrate magnetic particles within afluid without any external fluid transport mechanism that generateshydraulic pressure for fluid transport.

FIG. 13 shows a prototype system for transport of magnetic particles,the system comprising coil (inductor) array, switches and otherelectronic control elements, together with a prototype fluidic device(e.g., biochip). The prototype system of FIG. 13 was used to demonstratetransport of magnetic particles in the biochip, illustrated in FIG. 6.FIGS. 6 and 12 illustrate that the transport and/or concentration ofmagnetic particles demonstrated in FIG. 9 for a three coil array isscalable for any number coils.

FIG. 14 shows the specification for an embodiment of the prototypesystem of FIG. 13, indicating the magnetic coil structure and magneticfield strengths relative to the coil head surface. As one wouldrecognize, the magnetic field strengths would depend on the spacing ofthe coils, and the spacing could be varied in different electromagneticarrays.

Embodiments of the invention are directed to devices and methods fordetecting the presence of an analyte in a sample. According to oneembodiment, the device comprises a fluidic network comprising aplurality of fluidic zones, each fluidic zone being connected to theadjacent zone by a diffusion barrier, and an integrated circuitrycomponent. According to another embodiment, the invention encompasses adevice comprising a substrate containing a plurality of fluidic zones,each fluidic zone being connected to the adjacent fluidic zone by adiffusion barrier, wherein each fluidic zone comprises a fluid, andwherein one or more fluidic zones comprises a magnetic particle. In oneaspect, the device is adapted to permit transport of the magneticparticle from one fluidic zone to an adjacent fluidic zone substantiallywithout moving the fluid between the fluidic zones.

An array of magnetic microcoils can be functionally coupled to thefluidic network, which is programmably activatable to generate amagnetic field in proximity to each microcoil. The microcoil array canbe integrated into the network, or it can be located near the fluidiczones of the device, so that at least one microcoil is placed suitablyfor, generating a magnetic field in at least a portion of a fluidiczone. A detection element is also functionally coupled to the fluidicnetwork; it can be integrated into the network or located in proximityto the network. Generally, it is situated so that whether integrated ortemporarily coupled, it detects optical or electrical signals from oneor more of the fluidic zones. A vibration element can also befunctionally coupled to the network; it can be integrated into thenetwork or located in proximity to one or more fluidic zones. Typically,when activated, the vibration element is so situated that it willachieve the desired effect of shaking or agitating fluid within one ormore fluidic zones of the device.

Certain embodiments of the invention are self-contained such thatsubstantially no liquid flows between the fluidic zones, therebyeliminating the need for flow controllers. In such embodiments, themagnetic particles and any molecules bound to the magnetic particles aremoved through the liquid contained within the fluidic zones byactivating the magnetic microcoils, and are not moved by the flow of theliquid. Typically in these embodiments, the fluid is present in thefluidic zones to act as a suspending agent. Other embodiments of theinvention comprise a flow controller for coordinating liquid flowthrough the fluidic zones of the device. In such embodiments, themagnetic particles and any molecules bound to the magnetic particles aremoved through the fluidic zones by activating the magnetic microcoilsand/or also can be moved by activating the flow controller to move theliquid itself. The flow controller is functionally coupled to thenetwork: it can be integrated into the network or external to thenetwork.

The fluidic zones of the device typically comprise a reservoir, channel,groove, opening, or conduit in the substrate of the fluidic device,which is configured for containing a liquid and optionally forcontaining reagents. In one embodiment, the plurality of fluidic zonescomprises a sample zone, a cleaning zone and/or a detection zone. In afurther embodiment, it comprises more than one sample zone, cleaningzone, and/or detection zone. It can comprise additional fluidic zonesfor storing reagents, which can be branches of any of the aforementionedzones. In one embodiment, multiple fluidic zones are contained inparallel within the same device, thus allowing for analysis of multiplesamples or multiple analytes in parallel. Each fluidic zone is separatedfrom the adjacent fluidic zone by a diffusion barrier.

Diffusion barriers connect the fluidic zones of the device. They aredesigned and situated to minimize diffusion or convectance of thecontents of one fluidic zone to the next fluidic zone, such that themajority of the contents that move from one zone to the next fluidiczone are moved by directed fluidic flow and/or by activating themagnetic microcoil array. In certain embodiments, the diffusion barrieris a fluidic channel that is designed to alter the path of the fluidiczone. In other embodiments, the diffusion barrier is athermally-sensitive barrier. Hydrophilic fluid or liquid can becontained in a shape of droplets surrounded by hydrophobic liquid suchas silicone oils to form strong diffusion barriers throughhydrophilic-hydrophobic interactions so that droplets can be separatedand transported without mixing with other fluids as demonstrated in J.Micromech. Microeng. (2006) 16:1875 and Sensors and Actuators B (2006)113:563. A diffusion barrier can be accomplished by “particle trappingand transport” through DEP (dielectrophoresis) as demonstrated inBiophysical Journal (1998) 74:1024 and Sensors and Actuators A 121(2005) 59.

The detection element is situated in proximity to the detection zone.The detection element can be an optical detection element or anelectrical detection element. In certain embodiments, the opticaldetection element is selected from a Raman detector, a photon multipliertube, a fluorescent reader, or an electrochemical sensor and theelectrical detection element is selected from a FET element, a capacitydetection element, a current sensor, and a charge sensor. Typically, thedetection of the binding complex or the signal analyte complex indicatesthe presence of the analyte.

In further embodiments, the detection zone comprises a reactionsubstrate that interacts with a catalytic element to form a fluorogenic,chemiluminescent, or chromogenic product. Non-limiting examples ofreaction substrates include Lumigen APS-5, Lumigen TMA-6, LumigenPS-atto, Lumigen PS-3, H₂O₂ with an oxidizable compound, Amplex Red,3,5,3′,5′-tetramethylbenzidine (TMB), glucose, O₂, ATP, Mg²⁺, luciferin,inoluciferin, quinolinyl, coelentrazine, aldehyde, FMNH₂, and analogsand combinations thereof.

Typically, if the detection zone comprises a reaction substrate, themagnetic particle and/or the signal particle comprises a catalyticelement that serves as an agent to cause a chemical reaction to occur inthe reaction substrate, where the reaction product is detectable by thedetection element. In certain non-limiting embodiments, the catalyticelement is selected from the group consisting of alkaline phosphatase,horseradish peroxidase, glucose oxidase, luciferase (from firefly,Renilla, bacteria, or other sources) or analogs or combinations thereof.The catalytic element can be covalently or non-covalently conjugated tothe signal particle through a functionalized polymer. The fluidic zonesof the device generally contain an appropriate buffer to permit thereaction to occur.

The sample zone of the device comprises a magnetic particle selectedfrom the group consisting of a magnetic affinity complex and a codedmagnetic affinity complex. Magnetic particles may also be present withinother fluidic zones of the device. The microcoils are activated in sucha manner as to move the magnetic particles within the device.

The sample zone or other zone of the device can also comprise a signalparticle selected from the group consisting of signal affinitycomplexes, signal analyte complexes, and coded magnetic signal affinitycomplexes, among others. In certain embodiments the signal particle is aSERS-active nanoparticle, a fluorescent nanoparticle, a nanoparticlecoupled to a surface-enhanced fluorescent tag, or a core nanoparticlecovalently coupled to a catalytic element. In one embodiment, the signalparticle is a COIN particle. In other embodiments, the signal particleis a Qdot, or another fluorescent nanoparticle, such as SEF nanoparticleor a FluoDot. In further embodiments, the signal particle is anynanoparticle (i.e. gold, silver, CdS, CdSe, copper, Eu³⁺-coated polymer,an organic polymer (homo or hetero), an inorganic compound, or compositecompounds, etc.). Additionally, the SERS-active nanoparticle andfluorescent nanoparticle can also be functionally coupled to a catalyticelement. In certain embodiments, the sample zone of the fluidic devicecomprises the signal particle. Alternatively, the sample particle iscontained within another fluidic zone. In further embodiments, differentor the same signal particles can be contained within more than onefluidic zone.

Embodiments of the invention also include methods of using the devicesto detect the presence of an analyte.

The device contains magnetic particles within one or more fluidic zones,and the microcoil array is activated to thereby move the magneticparticles within that zone or to another zone. In one method, themagnetic particle within the sample zone is a magnetic affinity complex.A sample suspected of comprising an analyte is introduced into thesample zone, wherein the magnetic affinity complex binds to the analyteto form a magnetic binding complex. The microcoil array is activated tomove the magnetic binding complex from the sample zone to anotherfluidic zone.

In another embodiment, the magnetic particle is a magnetic signalaffinity complex. A sample suspected of comprising an analyte isintroduced into the sample zone, wherein the magnetic signal affinitycomplex binds to the analyte to form a magnetic signal binding complex.The microcoil array is activated to move the magnetic signal bindingcomplex from the sample zone to another fluidic zone. It is thendetected by the detection element, indicating the presence of theanalyte.

In another embodiment, one or more fluidic zones also comprise a signalaffinity complex. The analyte is combined with the magnetic affinitycomplex and the signal affinity complex, either simultaneously orsequentially, where the magnetic affinity complex and the signalaffinity complex bind to the analyte to form a sandwich binding complex.The microcoil array is activated to move the sandwich binding complex tothe detection zone of the fluidic network, where it is detected by thedetection element, and where the detection of the sandwich bindingcomplex indicates the presence of the analyte. In such an embodiment,the analyte is typically a protein, an antibody, or a nucleic acid.

In a further embodiment, the sample zone comprises a magnetic affinitycomplex, and one or more fluidic zones comprise a signal analytecomplex. The magnetic affinity complex binds to the analyte in thesample to form a magnetic binding complex. Optionally, the microcoilarray is activated to move the magnetic binding complex to anotherfluidic zone. The signal analyte complex then displaces the analyte fromthe magnetic binding complex to form a competitive binding complex.Optionally, the microcoil array is activated to move the competitivebinding complex to another fluidic zone. The detection element detectsan optical or electrical signal from the signal analyte complex that didnot form the competitive binding complex, thus indicating the presenceof the analyte. In such an embodiment, the analyte is typically a smallmolecule such as, but not limited to, sugars, drugs, steroids, andvitamins.

In another embodiment, the sample zone comprises a coded magneticaffinity complex. A sample suspected of comprising an analyte isintroduced a sample into the sample zone, wherein the coded magneticaffinity complex binds to the analyte to form a coded magnetic bindingcomplex. The microcoil array is activated to move the coded magneticbinding complex from the sample zone to a first affinity surface whereit is immobilized. Typically, the affinity agent of the first affinitysurface binds to the analyte or to the affinity agent coupled to themagnetic particle. The code is detached from the bound coded magneticbinding complex. A magnetic signal affinity complex is provided in oneof the fluidic zones, so situated that the detached code binds to themagnetic signal affinity complex to form a coded magnetic signal bindingcomplex. Typically, the affinity agent of the magnetic signal affinitycomplex is a polynucleotide complementary to the code. The microcoilarray is activated to move the coded magnetic signal binding complex tothe detection zone which comprises a second affinity surface, where itis immobilized. Typically, an affinity agent of the second affinitysurface comprises a polynucleotide complementary to the code. The codedmagnetic signal binding complex is then detected by the detectionelement. The second affinity surface can comprise an array of probes fordetecting any number of analytes.

The vibration element can be activated to agitate the fluid of one ormore of the fluidic zones. In certain embodiments, the vibration elementagitates the fluid in one or more fluidic zones to disperse the magneticparticles, analyte, and/or signal particles so that they can interact toform a binding complex. In other embodiments, the vibration elementagitates the fluid in one or more fluidic zones to facilitateaggregation-disaggregation and removal of unbound signal particlesand/or non-analyte components of the sample from the binding complex.For example, before the binding complex is moved to the detection zone,it is moved to the cleaning zone where the vibration element isactivated to aggregate and de-aggregate the binding complex to removeunbound signal particles and/or other components from the sample fromthe binding complex. In other embodiments, a coded magnetic bindingcomplex and/or a coded signal binding complex are moved to a cleaningzone by activating the microcoil array, wherein the vibration element isactivated to aggregate and de-aggregate the complexes to thereby removeunbound coded magnetic affinity complex, detached code, and/or magneticsignal affinity complex before the binding complex is moved to the nextzone.

Embodiments of the invention also comprise methods of fabricating thedevices. One embodiment comprises fabricating a plurality of fluidiczones on a substrate, where at least one of the fluidic zones is asample zone designed to hold a sample and a magnetic particle,fabricating one or more diffusion barriers on the substrate, wherein adiffusion barrier connects each fluidic zone to the adjacent fluidiczone; and forming an integrated circuitry component for storing data onthe substrate. The diffusion barrier can be fabricated as a fluidicchannel or as a thermally-sensitive barrier. In further embodiments, amicrocoil array is fabricated on the substrate. Alternatively, themicrocoil array is fabricated separately, and is removeably coupled tothe device when it is in use. A detection element can be fabricated intothe substrate, or can be fabricated separately and removeably coupled tothe device when in use. Preferably the detection element is an opticaldetection element or an electrical detection element. In furtherembodiments, a vibration element is fabricated into the device.Alternatively, the vibration element is fabricated separately andremoveably coupled to the device when in use.

In certain embodiments, fabricating the plurality of fluidic zones on asubstrate comprises combining two or more solid supports.

Embodiments of the invention also comprise a binding complex, which isan analyte bound to a magnetic affinity complex and a signal affinitycomplex. Typically the analyte is a protein, an antibody or a nucleicacid. In one embodiment, the analyte comprises an anti-PSA antibody. Ina further embodiment, the signal affinity complex comprises a COIN-PSAconjugate. The magnetic affinity complex can comprise astreptavidin-coated magnetic bead. The analyte can comprise an antibody,which includes an autoantibody.

As disclosed herein, compound and molecules suitable for analysis by theembodiments of the invention include proteins, peptides, and,specifically, nucleic acids (DNA and RNA), which can formdouble-stranded molecules by hybridization, that is, complementary basepairing. For example, in an embodiment of the invention, a molecularprobe, such as a DNA probe, is associated with or attached to a fluidiczone, which is located near or on the surface of, or otherwiseintegrated into, the substrate. The specificity of nucleic acidhybridization from the binding of the analyte to the molecular probe issuch that the detection of molecular and/or nanomaterials binding eventscan be done through measurements of the signals by the detection elementor other external circuitry. This specificity of complementary basepairing also allows thousands of hybridization to be carried outsimultaneously in the same experiment on a DNA chip (also called a DNAarray).

Molecular probes are immobilized on the surface of individual orindividually addressable reservoirs through surface functionalizationtechniques. The probe in a DNA chip is usually hybridized with a complexRNA or cDNA target (the analyte) generated by making DNA copies of acomplex mixture of RNA molecules derived from a particular cell type(source). The composition of such a target reflects the level ofindividual RNA molecules in the source. The optical or electricalsignals resulting from the binding events from the DNA spots of the DNAchip after hybridization between the probe and the target represent therelative expression levels of the genes of the source.

The DNA chip could be used for differential gene expression betweensamples (e.g., healthy tissue versus diseased tissue) to search forvarious specific genes (e.g., connected with an infectious agent) or ingene polymorphism and expression analysis. Particularly, the DNA chipcould be used to investigate expression of various genes connected withvarious diseases in order to find causes of these diseases and to enableaccurate treatments.

Using embodiments of the invention, one could find a specific segment ofa nucleic acid of a gene, i.e., find a site with a particular order ofbases in the examined gene. This detection could be performed by using adiagnostic polynucleotide made up of short synthetically assembledsingle-chained complementary polynucleotides—a chain of bases organizedin a mirror order to which the specific segment of the nucleic acidwould attach (hybridize) via A-T or G-C base pairing interactions.

The practice of the embodiments of the invention may employ, unlessotherwise indicated, conventional techniques of organic chemistry,polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the examples herein below.However, other equivalent conventional procedures can, of course, alsobe used.

The devices of the embodiments of the invention may be formed by anysuitable means of manufacture, including semiconductor manufacturingmethods, microforming processes, molding methods, material depositionmethods, etc., or any suitable combination of such methods. In certainembodiments one or more of the microcoils, and circuitries may be formedvia semiconductor manufacturing methods on a semiconductor substrate.Thin film coatings may be selectively deposited on portions of thesubstrate surface. Examples of suitable deposition techniques includevacuum sputtering, electron beam deposition, solution deposition, andchemical vapor deposition. The coatings may perform a variety offunctions. For example, the coatings may be used to increase thehydrophilicity of a surface or to improve high temperature properties.Conductive coatings may be used to form the microcoils. Coatings may beused to provide a physical barrier on the surface, e.g. to retain fluidat specific sites on the surface.

In one embodiment of the invention, the substrate is made throughcombining two or more smaller substrates or solid support. Specifically,the fabricating of the fluidic zones, or the fabricating of themicrocoils may involve combining two or more smaller substrates to formthe substrate.

The substrate used in the embodiments of the invention may comprisevarious materials including, but not limited to silicon, glass, metal,and polymeric material. According to the embodiments, the substratecomprises an integrated circuit, a microarray, a macroarray, fluidiczones, a detection element, a vibration element, or a combinationthereof.

In on embodiment of the invention, the sample zone for holding a samplecomprises a reservoir, a channel, an opening, a surface, or acombination thereof. According to another embodiment, the microcoilcomprises of copper, aluminum, gold, silver, or a mixture thereof. Themicrocoil is placed near or adjacent to the fluidic zones.

As disclosed herein, silicon is a suitable material for attaching othermaterials, such as metal or magnetic materials and forming structures,such as openings and channels coupled with microelectronics or othermicroelectromechanical systems (MEMS). It also has good stiffness,allowing the formation of fairly rigid microstructures, which can beuseful for dimensional stability. In a specific embodiment of theinvention, the substrate comprises an integrated circuitry componentselected from an integrated circuit (IC), a packaged integrated circuit,and an integrated circuit die. For example, the substrate may be apackaged integrated circuit that comprises a microprocessor, a networkprocessor, or other processing device.

In another embodiment, the method further comprises forming circuitry onor within the detection unit that is capable of amplifying or processingthe signals detected by the detection element. The substrate for thedetection element may be constructed using, for example, a ControlledCollapse Chip Connection (or “C4”) assembly technique, wherein aplurality of leads, or bond pads are internally electrically connectedby an array of connection elements (e.g., solder bumps, columns).

According to the embodiments of the invention, microcoils can befabricated on or within the substrate using a number of techniques,including etching, bonding, annealing, adhering/seeding, lithography,molding, and printing. Physical vapor deposition (PVD) and chemicalvapor deposition (CVD) can also be used. In one embodiment, microcoilsare fabricated on an oxidized silicon substrate by electroplating metalsinside a deep photoresist mold and then passivated using an epoxy basedresist.

The substrate of the embodiments of the present invention is suitablefor forming openings, voids, surfaces, or microchannels thereon forholding fluid and fluidic communications. The sample zone may be open orclosed along. Various methods may be used to form the sample zone on thesubstrate. For example, a reservoir or an open microchannel can befabricated on a silicon substrate by etching methods known to thoseskilled in the art. Closed channels can be formed by sealing the openchannels at top using methods such as anodic bonding of glass platesonto the open channels on the silicon substrate.

According to one embodiment of the invention, to fabricate a channel ona silicon substrate, a photoresist (positive or negative) is spun ontothe silicon substrate. The photoresist is exposed to UV light through ahigh-resolution mask with the desired device patterns. After washing offthe excessive unpolymerized photoresist, the silicon substrate is placedin a wet chemical etching bath that anisotropically etches the siliconin locations not protected by the photoresist. The result is a siliconsubstrate in which channels are etched. If desired, a glass cover slipis used to fully enclose the channels. Also, holes are drilled in theglass to allow fluidic access. For straighter edges and a deeper etchdepth, deep reactive ion etching (DRIE) can be used as an alternative towet chemical etching.

In another embodiment of the invention, channels may be formed on asilicon substrate using the following method. A seed layer of a metal,such as copper, is deposited over a surface of the substrate. Anysuitable blanket deposition process may be used to deposit the seedlayer of metal, such as physical vapor deposition (PVD), chemical vapordeposition (CVD), or other methods known to those skilled in the art. Alayer of a sacrificial material, such as a dielectric material or aphotoresist material, is then deposited over the seed layer. By removingthe sacrificial material, for example using chemical etch process orthermal decomposition process, a number of trenches in the sacrificiallayer are formed, and the seed layer is exposed in each of the trenches.Another layer of the metal, such as copper, is deposited over theexposed seed layer in the trenches. The metal layer extends overportions of the upper surface of the sacrificial layer; but gaps remainbetween the metal material layers extending from adjacent trenches andover the upper surface of the sacrificial layer. The sacrificial layeris removed, for example using chemical etching process or thermaldecomposition process, and regions from which the sacrificial layer hasbeen removed form channels in the metal layer. An additional layer ofthe metal is deposited over the upper surfaces of the metal layer toclose the gaps over the channels.

In the embodiments of the invention, reservoirs, openings and channelscan be made by using soft lithography method with suitable materials,such as silicon and polydimethylsiloxane (PDMS). With these techniquesit is possible to generate patterns with critical dimensions as small as30 nm. These techniques use transparent, elastomeric PDMS “stamps” withpatterned relief on the surface to generate features. The stamps can beprepared by casting prepolymers against masters patterned byconventional lithographic techniques, as well as against other mastersof interest. Several different techniques are known collectively as softlithography. They are as described below:

Near-Field Phase Shift Lithography. A transparent PDMS phase mask withrelief on its surface is placed in conformal contact with a layer ofphotoresist. Light passing through the stamp is modulated in thenear-field. Features with dimensions between 40 and 100 nm are producedin photoresist at each phase edge.

Replica Molding. A PDMS stamp is cast against a conventionally patternedmaster. Polyurethane is then molded against the secondary PDMS master.In this way, multiple copies can be made without damaging the originalmaster. The technique can replicate features as small as 30 nm.

Micromolding in Capillaries (MIMIC). Continuous channels are formed whena PDMS stamp is brought into conformal contact with a solid substrate.Capillary action fills the channels with a polymer precursor. Thepolymer is cured and the stamp is removed. MIMIC is able to generatefeatures down to 1 μm in size.

Microtransfer Molding ((TM). A PDMS stamp is filled with a prepolymer orceramic precursor and placed on a substrate. The material is cured andthe stamp is removed. The technique generates features as small as 250nm and is able to generate multilayer systems.

Solvent-assisted Microcontact Molding (SAMIM). A small amount of solventis spread on a patterned PDMS stamp and the stamp is placed on apolymer, such as photoresist. The solvent swells the polymer and causesit to expand to fill the surface relief of the stamp. Features as smallas 60 nm have been produced.

Microcontact Printing ((CP). An “ink” of alkanethiols is spread on apatterned PDMS stamp. The stamp is then brought into contact with thesubstrate, which can range from coinage metals to oxide layers. Thethiol ink is transferred to the substrate where it forms aself-assembled monolayer that can act as a resist against etching.Features as small as 300 nm have been made in this way.

Techniques used in other groups include micromachining of silicon formicroelectromechanical systems, and embossing of thermoplastic withpatterned quartz. Unlike conventional lithography, these techniques areable to generate features on both curved and reflective substrates andrapidly pattern large areas. A variety of materials could be patternedusing the above techniques, including metals and polymers. The methodscomplement and extend existing nanolithographic techniques and providenew routes to high-quality patterns and structures with feature sizes ofabout 30 nm.

Standard lithography on silicone wafer or silica glass could also beused to fabricate the devices of the embodiments of this invention.Reservoirs, openings and channels in the micrometer or nanometer scalecan be fabricated from the devices. If fluidic flow is employed, it canbe controlled by pressure gradient, electrical field gradient, gravity,and/or heat gradient. The surfaces of the fluidic zones and/or thediffusion barriers can be modified with polymers (polyethylene glycol(PEG)-dramatized compounds) that can minimize non-specific binding. Thesolid support can be inorganic material (e.g., glass, ceramic) or metal(e.g., aluminum). Biomolecules, proteins, antibodies, and/or nucleicacids can be coated on the surface of the substrate for specific analytebinding.

In the embodiments of the invention, the channels formed on thesubstrate may be straight or have angles or curves along their lengths.The characteristics and layout of the channels are determined by thespecific applications the device is designed for. Although straightchannels lining next to one another are a typical design formicrofluidic devices, the channels in the embodiments of the inventionmay be designed in many different patterns to serve specific separationand detection requirements. Specifically, the design of the channelstakes into consideration of the microcoils associated with the fluidiczones such that one or more microcoils are capable of generatingexcitation magnetic fields across at least a portion of one fluidiczones. Further, in the embodiments of the invention, the cross-sectionof the fluidic zone so formed may be uniform or vary along the channel'slength, and may have various shapes, such as rectangle, circle, orpolygon.

EXAMPLES Example 1 Magnetic Particles are Separated from SignalParticles in a Fluidic Device

A biochip was constructed as shown in FIG. 6, containing a sample zone,a cleaning zone and a detection zone, which was functionally coupled toa magnet. A mixture of magnetic particles and Qdot particles was loadedinto the sample zone. The arrows indicate the position of the magneticparticles over time, showing that they moved from the sample zone inpanel 1 to the detection zone in panel 6. UV fluorescence indicates thatthe Qdots were still located in the sample zone since no significantfluorescence was detected beyond the sample zone.

Solutions were retrieved from the sample zone and the detection zone,respectively, and finally adjusted to the same volumes for comparison.As a control, the same amount of particle mixture was cleaned in tubes;the supernatant from each step was saved to measure Qdot carry-over inthe absence of analyte (see FIG. 6B).

As shown by the control test in tubes (FIG. 6C), Qdots were separatedfrom magnetic particles after 4 washing steps; the same result could beachieved using the test chip; meaning the magnetic particles were freeof Qdots after they were transported from the sample zone to thedetection zone without liquid exchanges. For example, when PSA was theanalyte, as low as 0.1 pg of PSA was detected using SERS technology andCOIN particles (FIG. 6D).

The characteristics of some of the embodiments of the invention areillustrated in the Figures and examples, which are intended to be merelyexemplary of the invention. This application discloses several numericalrange limitations that support any range within the disclosed numericalranges even though a precise range limitation is not stated verbatim inthe specification because the embodiments of the invention could bepracticed throughout the disclosed numerical ranges. Finally, the entiredisclosure of the patents and publications referred in this application,if any, are hereby incorporated herein in entirety by reference.

We claim:
 1. A method comprising fabricating a fluidic networkcomprising a plurality of fluidic zones on a substrate, wherein at leastone of the fluidic zones is a sample zone designed to hold a sample anda magnetic particle, fabricating one or more diffusion barriers on thesubstrate, wherein a diffusion barrier connects each fluidic zone to theadjacent fluidic zone; forming an integrated circuitry component forstoring data on the substrate; and functionally connecting an array ofmagnetic microcoils to the substrate, wherein the magnetic microcoilsare programmably activatable to generate a magnetic field in proximityto each magnetic microcoil and to transport a magnetic particle in thefluidic network without fluidic movement of a fluid in the plurality ofthe fluidic zones.
 2. The method of claim 1, further comprisingfunctionally connecting a detection element to the substrate, whereinthe detection element is an optical detection element or an electricaldetection element.
 3. The method of claim 1, further comprisingfunctionally connecting a vibration element to the substrate, whereinthe vibration element is capable of vibrating a fluid within one or morefluidic zones.
 4. The method of claim 1, wherein fabricating theplurality of fluidic zones on a substrate comprises combining two ormore solid supports.
 5. The method of claim 1, wherein fabricating thediffusion barrier comprises fabricating a fluidic channel.
 6. The methodof claim 1, wherein fabricating the diffusion barrier comprisesfabricating a thermally-sensitive barrier.
 7. The method of claim 1,wherein the diffusion barrier is a hydrophilic-hydrophobic interface,created by completely surrounding the hydrophilic reagents byhydrophobic liquid.
 8. The method of claim 1, wherein the diffusionbarrier is created by a MEMS membrane valve.
 9. The method of claim 1,wherein the diffusion barrier is created by dielectric phoresis.
 10. Themethod of claim 1, wherein the plurality of fluidic zones comprises asample zone, a cleaning zone, and a detection zone.
 11. A methodcomprising providing a fluidic network comprising a plurality of fluidiczones, each fluidic zone being connected to the adjacent zone by adiffusion barrier, and an integrated circuitry component; functionallycoupling an array of magnetic microcoils to the fluidic network, whereinthe magnetic microcoils are programmably activatable to generate amagnetic field in proximity to each magnetic microcoil and to transporta magnetic particle in the fluidic network without fluidic movement of afluid in the plurality of the fluidic zones; functionally coupling adetection element to the fluidic network; and optionally coupling avibrational element to the fluidic network.
 12. The method of claim 11,wherein the plurality of fluidic zones comprises a sample zone, acleaning zone, and a detection zone; wherein the sample zone comprises aspace for holding a sample, and is selected from a reservoir, a channel,an opening, a surface, or a combination thereof.
 13. The method of claim12, wherein the sample zone comprises the magnetic particle selectedfrom the group consisting of a magnetic affinity complex, a competitivebinding complex and a coded magnetic affinity complex.
 14. The method ofclaim 13, wherein the array of magnetic microcoils is capable of movinga magnetic particle from the sample zone to the adjacent fluidic zone.15. The method of claim 11, wherein multiple sets of the plurality offluidic zones are provided in parallel in the fluidic network.
 16. Themethod of claim 11, wherein functionally coupling an array of magneticmicrocoils comprises removeably coupling the array of magneticmicrocoils to the fluidic network.
 17. The method of claim 11, whereinfunctionally coupling an array of magnetic microcoils comprisespermanently coupling the array of magnetic microcoils to the fluidicnetwork.
 18. The method of claim 11, wherein functionally coupling adetection element comprises selecting a detection element from anoptical detection element and an electrical detection element.
 19. Themethod of claim 16, wherein the optical detection element is selectedfrom, a photon multiplier tube, avalanche sensor, CCD, photodiode, aRaman detector, and a fluorescent reader, and the electrical detectionelement is selected from a capacity detection element, a current sensor,a voltage sensor, and a charge sensor, and an electrochemical sensor.20. The method of claim 11, wherein the device is formed to beself-contained.
 21. The method of claim 11, further comprisingfunctionally associating a flow controller with the fluidic network.